- Email: [email protected]
Effect of adenovirus-mediated TGF-β1 gene transfer on the function of rabbit articular chondrocytes
Journal of Orthopaedic Science xxx (2016) 1e7
Contents lists available at ScienceDirect
Journal of Orthopaedic Science journal homepage: http://www.elsevier.com/locate/jos
Original article
Effect of adenovirus-mediated TGF-b1 gene transfer on the function of rabbit articular chondrocytes Yuan Tang a, Jun Xiao a, Yeyang Wang b, Ming Li a, Zhanjun Shi a, * a b
Department of Orthopaedic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China Department of Orthopaedic Surgery, Third Affiliated Hospital of Southern Medical University, No. 183, Zhongshan West Road, Guangzhou 510631, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 May 2015 Received in revised form 18 March 2016 Accepted 18 May 2016 Available online xxx
Background: Articular chondrocytes are important in maintaining normal cartilage tissue and preventing articular degeneration. Exogenous genes have previously been transduced into articular cells using adenoviral vectors to contribute to the maintenance of cell function. This study aimed to transfer the transforming growth factor-b1 gene (TGF-b1) into rabbit articular chondrocytes by adenovirus infection to elucidate its effects on cell function. Methods: Rabbit chondrocytes were isolated and cultured both as monolayers and three-dimensional culture systems. To achieve overexpression, TGF-b1 was transfected by adenovirus infection, using the LacZ gene as a control. TGF-b1 protein expression was analyzed by western blotting. Quantitative DNA fluorometric analysis evaluated cell proliferation, and quantitative reverse transcriptase PCR determined the mRNA expression of related chondrocyte marker genes. Western blotting and glycosaminoglycan quantitative testing were used to examine changes in extracellular matrix components. Results: TGF-b1 protein expression was found to increase in Adv-TGF-b1-transduced cells, reaching a maximum after chondrocytes had been cultured for 4 weeks. Adv-hTGF-b1 transduction altered chondrocyte morphology from fibrocyte-like long spindle-shaped to round or oval. TGF-b1-transduced cells showed an increase in DNA synthesis, glycosaminoglycan content, and increased aggrecan and collagen II protein expression, while collagen I was significantly decreased. Moreover, TGF-b1 overexpression significantly promoted the mRNA expression of the chondrogenic gene SOX9, and inhibited that of the hypertrophic marker COL10A1 and the mineralization marker MMP-13. Conclusions: TGF-b1 overexpression positively improved the phenotype, function, and proliferation of chondrocytes, even after several generations. © 2016 The Japanese Orthopaedic Association. Published by Elsevier B.V. All rights reserved.
1. Introduction Cartilaginous tissue is mainly composed of chondrocytes and extracellular matrix (ECM), which is rich in matrix-associated water, collagens, and proteoglycans. Chondrocytes are essential for maintaining the ECM components, and chondrocyte dysfunction leads to cartilaginous degradation and corresponding changes in collagens and glycosaminoglycans (GAG), including chondroitin sulfate and keratan sulfate [1e3]. Extracellular environments play critical roles in maintaining original cell phenotypes, which can
* Corresponding author. Department of Orthopaedic Surgery, Nanfang Hospital, Southern Medical University, No. 1838 Guangzhou Dadaobei Avenue, Guangzhou 510515, China. Tel.: +86 15207729982. E-mail address: [email protected] (Z. Shi).
best be mimicked in vitro by a three-dimensional (3D) culture system [4,5]. Transforming growth factor-b1 gene (TGF-b1) is a 25 kDa polypeptide homodimer important in chondrocyte proliferation, differentiation, and extracellular matrix synthesis. The addition of TGF-b1 to chondrocyte cultures has previously been shown to be beneficial in inducing chondrocyte proliferation, maintaining phenotype stability, and inhibiting terminal differentiation and calcification [6,7]. Additionally, TGF-b1 increased the synthesis of ECM, including aggrecan and type II collagen, produced by chondrocytes and regulated cell expansion both in vitro and in vivo [8e10]. A previous study also showed that TGF-b1 prevented some inflammatory mediators (such as interleukin (IL)-1) from inhibiting the synthesis of proteoglycans in intervertebral discs, indicating that TGF-b1 may be suitable for cartilage repair. However, the biological half-life of TGF-b1 was found to be short when applied
http://dx.doi.org/10.1016/j.jos.2016.05.009 0949-2658/© 2016 The Japanese Orthopaedic Association. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Tang Y, et al., Effect of adenovirus-mediated TGF-b1 gene transfer on the function of rabbit articular chondrocytes, Journal of Orthopaedic Science (2016), http://dx.doi.org/10.1016/j.jos.2016.05.009
2
Y. Tang et al. / Journal of Orthopaedic Science xxx (2016) 1e7
directly, and it is not clear how to maintain a high concentration of TGF-b1 in cultured cells. Exogenous genes have been efficiently transferred into intervertebral disc cells using adenoviral vectors. The advantages of adenovirus-mediated gene delivery are that it is not influenced by the cell cycle, a large number of cells can accept multiple copies of recombinant adenovirus DNA, and transferred genes show prolonged expression during in vitro culture [11e13]. Therefore, in the present study, we attempted to transfer TGF-b1 using an adenoviral vector to achieve high concentrations in the matrix, and to investigate the effects of Adv-hTGF-b1 on chondrocyte function and ECM components. We established a recombinant hTGF-b1 overexpression system, and detected the effects of TGF-b1 on the regulation of type II collagen and aggrecan in a monolayer culture system and a 3D calciumealginate gel matrix culture system. This study assessed the application of TGF-b1 for the biological repair of cartilage. 2. Materials and methods
2.4. Calciumealginate gel bead culture A total of 2 106 chondrocytes were suspended in 1 ml 1.2% sodium alginate solution, then mixed with 102 mmol/L CaCl2 solution for 15 min without shaking, to form calciumealginate gel beads. Excess liquid was removed, and gel beads were washed three times using normal saline and DMEM medium. Ten beads were placed in each well of a 24-well tissue culture plate (Falcon) and cultured in 2 ml Ham's F12 medium supplemented with 10% FBS for 10 days. 2.5. Cell morphological analysis After culture in monolayers and 3D calciumealginate gel matrix culture systems for 10 days, the morphology of chondrocytes with or without Adv-hTGF-b1 transduction was examined by staining with the hematoxylin and eosin (HE) kit (Solarbio) or Alcian Blue kit (Shanghai yuanye Bio-Technology Co., Ltd) according to the manufacturer's instructions.
2.1. Chondrocyte isolation and culture
2.6. Quantitative fluorometric DNA analysis
The knee cartilage of New Zealand white rabbits was obtained from the tibial platform and the femoral condyle, in accordance with protocols approved by the Animal Care Committee of the Southern Medical University. Cartilage pieces (approximately 1 mm3) were treated with trypsin and type II collagenase (Sigma) overnight. Cells were then cultured in Ham's F12 medium supplemented with 10% fetal bovine serum (FBS, GIBCO-BRL) at 37 C with 5% CO2. The culture medium was refreshed every 2 days. The third generation of chondrocytes was used for the following study.
DNA content was evaluated by the bisbenzimidazole fluorescent dye assay. Briefly, 10 days after transfection, chondrocytes were digested in papain solution (125 mg/ml papain in phosphatebuffered saline, 5 mmol/L Na2EDTA, pH 6.0) and then reacted with 0.1 mg/ml bisbenzimidazole fluorescent dye in 10 mmol/L TriseHCl, pH 7.4. The DNA concentration was measured by a microplate reader with an excitation of 365 nm and an emission of 400e550 nm. 2.7. Quantitative RT-PCR
2.2. Recombinant adenoviral vector construction An adenoviral vector encoding TGFb1 (Adv-TGFb1) and a control null adenovirus (Adv-LacZ) were constructed as previously described [14,15]. First, Adv-TGFb1 mRNA was isolated from MCF10A cells, then cloned into a shuttle plasmid and cotransfected into human embryonic kidney 293 cells (ATCC, Rockville, MD) to generate the adenoviral vector. Adenoviral products were purified by CsCl gradient centrifugation and PD-10 Sephadex chromatog Que , Canada), then raphy (Amersham Pharmacia, Baie d'Urfe titered by plaque assays on the same 293 cells. Purified Adv-TGFb1 and Adv-LacZ adenoviruses were transfected into rabbit articular chondrocytes (see below), at which point the TGFb1 fragment was expressed as LAP-TGFb1. LAP was then cleaved off by protease furin in the Golgi apparatus, and finally excreted out of the cell [16]. All experimental protocols followed the Biosafety Committee of McMaster University (AUP-060313). 10 days later, supernatants were harvested and analyzed by enzyme-linked immunosorbent assays (ELISA; R&D Systems, Minneapolis, Min., USA) confirming TGFb1 overexpression. 2.3. Adv-hTGF-b1-transduced culture Chondrocytes were transfected with 200 multiplicity of infection of Adv-hTGF-b1 for 8 h, then the medium was changed to fresh Ham's F12 medium supplemented with 10% FBS. Primary untransduced chondrocytes and Lac-Z-transduced chondrocytes served as controls. Supernatants were harvested 10 days later and analyzed by enzyme-linked immunosorbent assays (R&D Systems, Minneapolis, MN) to detect the expression of TGFb1 and relevant ECM proteins.
After being cultured in monolayers and 3D calciumealginate gel matrix culture systems for 10 days, chondrocytes underwent total RNA extraction using the TriPure Isolation Reagent (Invitrogen Corp.). RNA was quantified by ultraviolet spectroscopy (Thermo Scientific), then 1 mg was reverse-transcribed into cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science). PCR amplification was performed using a Light Cycler Instrument and the Light Cycler Fast Start DNA Master SYBR Green 1 Kit (Roche Applied Science) according to the manufacturer's instructions. Finally, gene expression was quantified relatively by ABI Prism 7700 Sequence Detection System as a fold-increase compared with primary chondrocytes. Primer sequences are listed in Table 1. 2.8. Western blotting analysis Protein expression of TGF-b1, aggrecan, collagen-I, and collagenII were evaluated by western blotting after chondrocytes had been cultured for 10 and 36 days. Briefly, proteins were extracted from harvested chondrocytes and then 20 mL of protein was separated on 10% sodium dodecyl sulfate-polyacrylamide gels before being transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% dried milk at room temperature for 60 min. They were then incubated at 4 C overnight on a shaking platform with primary antibodies anti-b-actin (rabbit monoclonal antibody, SigmaeAldrich), anti-aggrecan (rabbit polyclonal antibody, SigmaeAldrich), anti-Col I (mouse monoclonal antibody, SigmaeAldrich), and anti-Col II (mouse monoclonal antibody, Invitrogen Corp.). Signals were detected using the enhanced chemiluminescence kit (Perkin Elmer, Waltham, MA) and quantified by the computer program ‘Quantity one’ (Bio-Rad).
Please cite this article in press as: Tang Y, et al., Effect of adenovirus-mediated TGF-b1 gene transfer on the function of rabbit articular chondrocytes, Journal of Orthopaedic Science (2016), http://dx.doi.org/10.1016/j.jos.2016.05.009
Y. Tang et al. / Journal of Orthopaedic Science xxx (2016) 1e7
3
Table 1 Primer sequences used for RT-qPCR. Gene
Forward
Reverse
SOX9 COL10A1 MMP13 b-actin
50 -ACCTCAAGAAGGAGAGCGAAG-30 50 -GGCTTCCCAGTGGCTGATAG-30 50 -GAATTAAGGAGCATGGCG AC-30 50 -AGACCACCTTCAACTCGATCAT-30
50 -CACCAGCGTCCAGTCGTAG-30 50 -TTTTGCTCTCTCTGGGTGGC-30 50 -TAAGGAGTGGCCGAACTCAT-30 50 -ACTCGTCATACTCCTGCTTGCT-30
2.9. Measurement of GAG contents After being cultured for 10 days, chondrocytes were treated using cell lysis buffer mixed with Alcian Blue solution (pH 2.0) to evaluate chronogenesis. They were washed with 40% dimethyl sulfoxide and 0.05 mol/L MgCl2, and dissolved in 4.0 mol/L guanidineeHCl and 33% propanol. The optical density of the extracted dye was measured on a 96-well plate reader at 600 nm.
several generations demonstrated long spindle-shaped fibrocytelike adherent growth. Adv-TGF-b1-transduced chondrocytes (T) showed a similar morphology to primary chondrocytes. We observed similar results following chondrocyte staining with HE and Alcian Blue. Chondrocytes cultured with 3D calciumealginate gel beads maintained their morphology, and almost no morphological changes were found among the four groups. 3.2. Analysis of TGF-b1 expression
2.10. Statistical analysis All data are presented as means ± SD and analyzed with SPSS 13.0 software. Comparisons were processed by one-way analysis of variance with p < 0.05 considered significant. 3. Results
Fig. 2A shows TGF-b1 expression levels in cells cultured for different times. TGF-b1 expression gradually increased over time up to the point at which chondrocytes had been cultured for 27 days, after which it declined. As shown in Fig. 2B, C, TGF-b1 expression was maintained at relatively low levels in Pri, NC, and BC groups, but was markedly increased in Adv-hTGF-b1-transduced chondrocytes in both culture systems after several generations.
3.1. Phenotypic analysis of chondrocytes in each culture system 3.3. Overexpression of TGF-b1 promotes DNA synthesis The morphology of chondrocytes cultured in monolayers and the 3D culture system is shown in Fig. 1. Primary chondrocytes (Pri) were shown to be round or oval, while Adv-LacZ transduced chondrocytes (NC) and blank control (BC) groups cultured for
The chondrocyte DNA content was determined by quantitative fluorometric analysis. In the monolayer culture system, TGF-b1 was found to promote DNA synthesis in chondrocytes compared with
Fig. 1. Morphological changes of chondrocytes cultured in both culture systems for 10 days were examined under a light microscope. Chondrocyte monolayers were stained with hematoxylin and eosin (HE) and Alcian Blue. Cells from 3D culture were captured using bright-field imaging without staining. Pri: primary chondrocytes; T: Adv-TGF-b1-transduced chondrocytes; NC: Adv-LacZ-transduced chondrocytes; BC: blank control (magnification, 100).
Please cite this article in press as: Tang Y, et al., Effect of adenovirus-mediated TGF-b1 gene transfer on the function of rabbit articular chondrocytes, Journal of Orthopaedic Science (2016), http://dx.doi.org/10.1016/j.jos.2016.05.009
4
Y. Tang et al. / Journal of Orthopaedic Science xxx (2016) 1e7
Fig. 2. Western blotting analysis of TGF-b1 expression in chondrocytes cultured for up to 36 days (A), for 10 days (B), and densitometric analysis (C). Data are mean ± SD of three experiments, *p < 0.05.
3.4. Effects of TGF-b1 on aggrecan and GAG levels Fig. 4 shows the absorbance value of GAG in chondrocyte supernatants. We found that this value was markedly higher in AdvhTGF-b1-transduced chondrocytes than in Adv-LacZ-transduced chondrocytes in both the monolayer and 3D culture systems. Moreover, western blot analysis revealed a similar trend of aggrecan expression with GAG synthesis, as shown in Fig. 5. A significant correlation was detected between the two indices, while aggrecan and GAG expression levels were relatively higher in the 3D culture system compared with the monolayer culture system.
Fig. 3. DNA content in rabbit chondrocytes cultured for 10 days as determined by quantitative DNA fluorometric analysis. Each data point represents the mean ± SD from three independent experiments, *p < 0.05.
LacZ transduction (Fig. 3). No significant difference was observed between Adv-LacZ-transduced chondrocytes and the BC group or between Adv-hTGF-b1-transduced chondrocytes and primary chondrocytes. In the 3D culture system, no significant difference was observed among all groups, which was consistent with the cellular morphology findings.
Fig. 4. Sulfated glycosaminoglycan (GAG) content in chondrocyte supernatants after being cultured for 10 days. Each data point represents the mean ± SD from three independent experiments, *p < 0.05.
3.5. Gene expression of chondrocytes RT-qPCR was used to assess gene expression of chondrocytes cultured in monolayers and the 3D culture system. As shown in Fig. 6, SOX9 mRNA expression was significantly decreased in NC and BC groups in both culture systems, but almost recovered to primary chondrocyte levels after Adv-hTGF-b1 transduction.
Fig. 5. Effect of gene delivery on aggrecan (Agn) expression in chondrocytes cultured for 10 days. (A) Agn protein levels were analyzed by western blot. (B) Densitometric analysis of Agn. Data points represent means ± SD from three independent experiments, *p < 0.05.
Please cite this article in press as: Tang Y, et al., Effect of adenovirus-mediated TGF-b1 gene transfer on the function of rabbit articular chondrocytes, Journal of Orthopaedic Science (2016), http://dx.doi.org/10.1016/j.jos.2016.05.009
Y. Tang et al. / Journal of Orthopaedic Science xxx (2016) 1e7
5
Fig. 6. RT-qPCR analysis of chondrocytes cultured in monolayer and 3D culture systems for 10 days. The induction of gene expression was determined relative to primary chondrocytes. Each data point represents the mean ± SD from three independent experiments, *p < 0.05.
Expression of the hypertrophic/mineralization markers COL10A1 and matrix metalloproteinase-13 (MMP13) were increased in NC and BC groups, but there were no significant differences between the T group and Pri group. These findings suggest that Adv-hTGF-b1 transduction promotes SOX9 mRNA expression and inhibits the expression of COL10A1 and MMP13 in monolayers and 3D culture systems.
3.6. Western blot analysis of type I and II collagen As shown in Fig. 7, western blot analysis revealed that primary chondrocytes mainly produced type II collagen (Fig. 7A, B) and low levels of type I collagen (Fig. 7A, C). The levels of type II collagen reduced and those of type I collagen increased after several generations in both culture systems. However, the trends of collagen expression reversed after Adv-hTGF-b1 transfection (Fig. 7B, C). Additionally, the ratio of expression between type II and type I collagen approached that seen in primary chondrocytes, especially in the 3D culture system (Fig. 7D). Taken together, these observations suggest that TGF-b1 maintained type II collagen expression in chondrocytes.
4. Discussion Previous cures for articular cartilage degeneration have mainly focused on autologous chondrocyte implantation, tissueengineering product replacement, and ex vivo gene therapy [17,18]. However, many issues still exist regarding these methods; for example, chondrocytes lose their original phenotype when cultured in a monolayer culture system. The delivery of an exogenous gene and the regulation of target cell function are also
difficult. One solution may be to overexpress a growth factor in autologous cells through genetic regulation. The reduction of chondrocyte activity and changes to cartilage ECM components always occur during subculture, so an appropriate chondrocyte phenotype is very important for its clinical application [19]. Within articular cartilage, the ECM plays a crucial role in regulating chondrocyte functions and metabolism. High levels of aggrecan and type II collagen are primary components of ECM [20]. In the present study, we found that primary chondrocytes cultured in vitro produce high levels of aggrecan and type II collagen, which decreases significantly after several generations of culture. The original primary chondrocyte phenotype also changed and its activity degraded after several generations in vitro. TGF-b1, as a member of the TGF-b superfamily, is a critical regulator of the development and differentiation of bone tissue. It regulates the production of collagen and aggrecan, and promotes bone marrow stromal cell (BMSC) proliferation and differentiation [21]. Tougu Xiaotong formula was reported to induce chondrogenic differentiation of BMSCs in association with TGF-b1 by activating the Ihh signaling pathway [22], while TGF-b1 expression in genetically modified human chondrocytes was proven to sustain positive effects on functional outcomes and cartilage growth in initial trials [23]. These studies demonstrate that TGF-b1 plays an important role in the development and maintenance of articular cartilage [24]. In the current study, we found that the expression of exogenous TGF-b1 was maintained after chondrocyte culture in monolayers and 3D culture systems for more than 1 month, with maximum levels reached after culture for 27 days. In the absence of exogenous TGF-b1, chondrocyte proliferation slowed down, and the cells resembled fibroblasts and eventually underwent apoptosis after in vitro culture. However, TGF-b1 overexpression through
Please cite this article in press as: Tang Y, et al., Effect of adenovirus-mediated TGF-b1 gene transfer on the function of rabbit articular chondrocytes, Journal of Orthopaedic Science (2016), http://dx.doi.org/10.1016/j.jos.2016.05.009
6
Y. Tang et al. / Journal of Orthopaedic Science xxx (2016) 1e7
Fig. 7. Col-I and col-II protein expression in chondrocytes cultured for 10 days. (A) col-I and col-II protein levels. Densitometric analysis of col-II (B) and col-I (C). (D) Ratio of col-II/ col-I protein expression. Data are mean ± SD of three experiments, *p < 0.05.
adenoviral transduction prevented the degradation of rabbit articular chondrocytes. TGF-b1 not only significantly promoted cell division and DNA synthesis, but also enhanced the synthesis of GAG and type II collagen. SOX9 is a chondrogenic transcription factor involved in chondrocyte differentiation and the expression of chondrocyte-specific anabolic genes such as type II collagen and aggrecan [25e27]. COL10A1 and MMP-13 have been widely interpreted as markers of hypertrophic or “osteoarthritic” chondrocytes, with COL10A1 an indicator of endochondral ossification, and MMP13 found to interfere with ECM synthesis [28,29]. In the present study, we found that expression of SOX9 was enhanced and that of COL10A1 and MMP-13 was inhibited after Adv-hTGF-b1 transduction of chondrocytes. Therefore, Adv-hTGF-b1 was beneficial in maintaining the phenotype of chondrocytes. The 3D calciumealginate culture system, as one of the best in vitro culture models, has been demonstrated to provide an environment close to the in vivo conditions necessary for assessing the functional status of nucleus pulposus cells [30]. To eliminate the influence of ECM components and tissue microenvironments in our study, chondrocytes were cultured within alginate gel beads in all groups. Although Adv-hTGF-b1-transduced chondrocytes expressed high levels of TGF-b1, DNA synthesis did not increase in cells grown in the 3D culture, indicating that there was no significant difference in cell proliferation between the TGF-b1-transduced group and control groups. However, the expression levels of aggrecan and type II collagen increased significantly after TGF-b1 transfection. The biological effect of TGF-b1 on chondrocytes may relate to certain temporary factors. Because the third generation of chondrocytes cultured in the 3D calciumealginate culture system
showed a similar phenotype to primary chondrocytes, this demonstrates that TGF-b1 released by gels gradually accumulated in Adv-hTGF-b1-transduced chondrocytes, then stimulated ECM synthesis and regulated the balance of ECM components. Thus, Adv-hTGF-b1-transduced chondrocytes appear to be a good prospect for re-implantation into degenerative joints. Moreover, the increased expression of ECM proteins regulated by TGF-b1 would be likely to recover the original environments surrounding articular chondrocytes and reverse articular degeneration. Furthermore, Adv-hTGF-b1-transduced articular chondrocytes should achieve a stable high concentration of TGF-b1 with no side-effects to adjacent tissue after transplantation. In conclusion, the overexpression of TGF-b1 was shown to be beneficial in improving the phenotype and function of chondrocytes. Therefore, TGF-b1 represents a potential therapeutic target for the modulation of cartilage development. However, further studies should be performed to investigate mechanisms of selective TGF-b1 targeting. Conflicts of interest All authors have seen and agree with the content of the manuscript and there is no conflict of interest to the report. References [1] Espanha MM. Articular cartilage: structure and histochemical composition. Acta Reumatol Port 2010 OcteDec;35(5):424e33. [2] Maldonado M, Nam J. The role of changes in extracellular matrix of cartilage in the presence of inflammation on the pathology of osteoarthritis. Biomed Res Int 2013;2013:284873.
Please cite this article in press as: Tang Y, et al., Effect of adenovirus-mediated TGF-b1 gene transfer on the function of rabbit articular chondrocytes, Journal of Orthopaedic Science (2016), http://dx.doi.org/10.1016/j.jos.2016.05.009
Y. Tang et al. / Journal of Orthopaedic Science xxx (2016) 1e7 [3] Hunziker EB, Lippuner K, Shintani N. How best to preserve and reveal the structural intricacies of cartilaginous tissue. Matrix Biol 2014 Oct;39:33e43. [4] Kim IL, Mauck RL, Burdick JA. Hydrogel design for cartilage tissue engineering: a case study with hyaluronic acid. Biomaterials 2011 Dec;32(34):8771e82. [5] Schagemann JC, Mrosek EH, Landers R, Kurz H, Erggelet C. Morphology and function of ovine articular cartilage chondrocytes in 3-D hydrogel culture. Cells Tissues Organs 2006 Jun;182(2):89e97. [6] Xu HG, Hu CJ, Wang H, Liu P, Yang XM, Zhang Y, Wang LT. Effects of mechanical strain on ANK, ENPP1 and TGF-beta1 expression in rat endplate chondrocytes in vitro. Mol Med Rep 2011 SepeOct;4(5):831e5. [7] Goldberg AJ, Lee DA, Bader DL, Bentley G. Autologous chondrocyte implantation. Culture in a TGF-beta-containing medium enhances the re-expression of a chondrocytic phenotype in passaged human chondrocytes in pellet culture. J Bone Jt Surg Br 2005 Jan;87(1):128e34. [8] Chen S, Emery SE, Pei M. Coculture of synovium-derived stem cells and nucleus pulposus cells in serum-free defined medium with supplementation of transforming growth factor-beta1: a potential application of tissue-specific stem cells in disc regeneration. Spine (Phila Pa 1976) 2009 May;34(12): 1272e80. [9] Liu H, Ning B, Wang D, Hu Y. Biological effects of recombinant adenoassociated virus 2 mediated human transforming growth factor beta1, encoding gene transfer to rabbit degenerative nucleus pulposus cells on proteoglycan level. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi 2010 May;24(5):618e21. [10] Li X, Hou X, Tang T. Increased synthesis of extracellular matrix in passaged nucleus pulposus cells by transfection with adenoviral vectors containing human transforming growth factor beta1. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi 2007 Dec;21(12):1342e7. [11] Zacchigna S, Zentilin L, Giacca M. Adeno-associated virus vectors as therapeutic and investigational tools in the cardiovascular system. Circulation Res 2014 May;114(11):1827e46. [12] Wold WSM, Toth K. Adenovirus vectors for gene therapy, vaccination and cancer gene therapy. Curr Gene Ther 2013 Dec;13(6):421e33. [13] Crystal RG. Adenovirus: the first effective in vivo gene delivery vector. Hum Gene Ther 2014 Jan;25(1):3e11. [14] Sime PJ, Xing Z, Graham FL, Csaky KG, Gauldie J. Adenovector-mediated gene transfer of active transforming growth factor-beta1 induces prolonged severe fibrosis in rat lung. J Clin Investig 1997 Aug;100(4):768e76. [15] Bett AJ, Haddara W, Prevec L, Graham FL. An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3. Proc Natl Acad Sci U S A 1994 Sep;91(19):8802e6. [16] Tritschler I, Gramatzki D, Capper D, Mittelbronn M, Meyermann R, Saharinen J, Wick W, Keski-Oja J, Weller M. Modulation of TGF-beta activity by latent TGF-beta-binding protein 1 in human malignant glioma cells. Int J Cancer 2009 Aug 1;125(3):530e40. [17] Makris EA, Gomoll AH, Malizos KN, Hu JC, Athanasiou KA. Repair and tissue engineering techniques for articular cartilage. Nat Rev Rheumatol 2015 Jan;11(1):21e34.
7
[18] Musumeci G, Castrogiovanni P, Leonardi R, Trovato FM, Szychlinska MA, Di Giunta A, Loreto C, Castorina S. New perspectives for articular cartilage repair treatment through tissue engineering: a contemporary review. World J Orthop 2014 Apr;5(2):80e8. [19] Ito A, Aoyama T, Iijima H, Tajino J, Nagai M, Yamaguchi S, Zhang X, Kuroki H. Culture temperature affects redifferentiation and cartilaginous extracellular matrix formation in dedifferentiated human chondrocytes. J Orthop Res 2015 May;33(5):633e9. [20] Gao Y, Liu S, Huang J, Guo W, Chen J, Zhang L, Zhao B, Peng J, Wang A, Wang Y, Xu W, Lu S, Yuan M, Guo Q. The ECMecell interaction of cartilage extracellular matrix on chondrocytes. Biomed Res Int 2014;2014:648459. [21] Miura Y, Parvizi J, Fitzsimmons JS, O'Driscoll SW. Brief exposure to high-dose transforming growth factor-beta1 enhances periosteal chondrogenesis in vitro: a preliminary report. J Bone Jt Surg Am 2002 May;84-A(5):793e9. [22] Chen J, Liu G, Weng X, Liu F, Lin P, Li H, Chen W, Huang Y, Liu X, Ye H, Li X. Tougu Xiaotong formula induces chondrogenic differentiation in association with transforming growth factor-beta1 and promotes proliferation in bone marrow stromal cells. Int J Mol Med 2015 Mar;35(3):747e54. [23] Elmallah RK, Cherian JJ, Jauregui JJ, Pierce TP, Beaver WB, Mont MA. Genetically modified chondrocytes expressing TGF-beta1: a revolutionary treatment for articular cartilage damage? Expert Opin Biol Ther 2015 Mar;15(3):455e64. [24] Huang X, Xu J, Huang M, Li J, Dai L, Dai K, Zhang X. Histone deacetylase1 promotes TGF-beta1-mediated early chondrogenesis through downregulating canonical Wnt signaling. Biochem Biophys Res Commun 2014 Oct;453(4):810e6. [25] de Crombrugghe B, Lefebvre V, Behringer RR, Bi W, Murakami S, Huang W. Transcriptional mechanisms of chondrocyte differentiation. Matrix Biol 2000 Sep;19(5):389e94. [26] Kulyk WM, Franklin JL, Hoffman LM. Sox9 expression during chondrogenesis in micromass cultures of embryonic limb mesenchyme. Exp Cell Res 2000 Mar;255(2):327e32. [27] Seifarth C, Csaki C, Shakibaei M. Anabolic actions of IGF-I and TGF-beta1 on interleukin-1beta-treated human articular chondrocytes: evaluation in two and three dimensional cultures. Histol Histopathol 2009 Oct;24(10):1245e62. [28] Caron MMJ, Emans PJ, Coolsen MME, Voss L, Surtel DAM, Cremers A, van Rhijn LW, Welting TJM. Redifferentiation of dedifferentiated human articular chondrocytes: comparison of 2D and 3D cultures. Osteoarthr Cartil 2012 Oct;20(10):1170e8. [29] Plaas A, Velasco J, Gorski DJ, Li J, Cole A, Christopherson K, Sandy JD. The relationship between fibrogenic TGFb1 signaling in the joint and cartilage degradation in post-injury osteoarthritis. Osteoarthr Cartil 2011 Sep;19(9): 1081e90. [30] Callejas FD, Martinez-Anton A, Alobid I, Fuentes M, Cortijo J, Picado C, RocaFerrer J, Mullol J. Reconstituted human upper airway epithelium as 3-D in vitro model for nasal polyposis. PLoS One 2014 Jun;9(6).
Please cite this article in press as: Tang Y, et al., Effect of adenovirus-mediated TGF-b1 gene transfer on the function of rabbit articular chondrocytes, Journal of Orthopaedic Science (2016), http://dx.doi.org/10.1016/j.jos.2016.05.009
Contents lists available at ScienceDirect
Journal of Orthopaedic Science journal homepage: http://www.elsevier.com/locate/jos
Original article
Effect of adenovirus-mediated TGF-b1 gene transfer on the function of rabbit articular chondrocytes Yuan Tang a, Jun Xiao a, Yeyang Wang b, Ming Li a, Zhanjun Shi a, * a b
Department of Orthopaedic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China Department of Orthopaedic Surgery, Third Affiliated Hospital of Southern Medical University, No. 183, Zhongshan West Road, Guangzhou 510631, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 May 2015 Received in revised form 18 March 2016 Accepted 18 May 2016 Available online xxx
Background: Articular chondrocytes are important in maintaining normal cartilage tissue and preventing articular degeneration. Exogenous genes have previously been transduced into articular cells using adenoviral vectors to contribute to the maintenance of cell function. This study aimed to transfer the transforming growth factor-b1 gene (TGF-b1) into rabbit articular chondrocytes by adenovirus infection to elucidate its effects on cell function. Methods: Rabbit chondrocytes were isolated and cultured both as monolayers and three-dimensional culture systems. To achieve overexpression, TGF-b1 was transfected by adenovirus infection, using the LacZ gene as a control. TGF-b1 protein expression was analyzed by western blotting. Quantitative DNA fluorometric analysis evaluated cell proliferation, and quantitative reverse transcriptase PCR determined the mRNA expression of related chondrocyte marker genes. Western blotting and glycosaminoglycan quantitative testing were used to examine changes in extracellular matrix components. Results: TGF-b1 protein expression was found to increase in Adv-TGF-b1-transduced cells, reaching a maximum after chondrocytes had been cultured for 4 weeks. Adv-hTGF-b1 transduction altered chondrocyte morphology from fibrocyte-like long spindle-shaped to round or oval. TGF-b1-transduced cells showed an increase in DNA synthesis, glycosaminoglycan content, and increased aggrecan and collagen II protein expression, while collagen I was significantly decreased. Moreover, TGF-b1 overexpression significantly promoted the mRNA expression of the chondrogenic gene SOX9, and inhibited that of the hypertrophic marker COL10A1 and the mineralization marker MMP-13. Conclusions: TGF-b1 overexpression positively improved the phenotype, function, and proliferation of chondrocytes, even after several generations. © 2016 The Japanese Orthopaedic Association. Published by Elsevier B.V. All rights reserved.
1. Introduction Cartilaginous tissue is mainly composed of chondrocytes and extracellular matrix (ECM), which is rich in matrix-associated water, collagens, and proteoglycans. Chondrocytes are essential for maintaining the ECM components, and chondrocyte dysfunction leads to cartilaginous degradation and corresponding changes in collagens and glycosaminoglycans (GAG), including chondroitin sulfate and keratan sulfate [1e3]. Extracellular environments play critical roles in maintaining original cell phenotypes, which can
* Corresponding author. Department of Orthopaedic Surgery, Nanfang Hospital, Southern Medical University, No. 1838 Guangzhou Dadaobei Avenue, Guangzhou 510515, China. Tel.: +86 15207729982. E-mail address: [email protected] (Z. Shi).
best be mimicked in vitro by a three-dimensional (3D) culture system [4,5]. Transforming growth factor-b1 gene (TGF-b1) is a 25 kDa polypeptide homodimer important in chondrocyte proliferation, differentiation, and extracellular matrix synthesis. The addition of TGF-b1 to chondrocyte cultures has previously been shown to be beneficial in inducing chondrocyte proliferation, maintaining phenotype stability, and inhibiting terminal differentiation and calcification [6,7]. Additionally, TGF-b1 increased the synthesis of ECM, including aggrecan and type II collagen, produced by chondrocytes and regulated cell expansion both in vitro and in vivo [8e10]. A previous study also showed that TGF-b1 prevented some inflammatory mediators (such as interleukin (IL)-1) from inhibiting the synthesis of proteoglycans in intervertebral discs, indicating that TGF-b1 may be suitable for cartilage repair. However, the biological half-life of TGF-b1 was found to be short when applied
http://dx.doi.org/10.1016/j.jos.2016.05.009 0949-2658/© 2016 The Japanese Orthopaedic Association. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Tang Y, et al., Effect of adenovirus-mediated TGF-b1 gene transfer on the function of rabbit articular chondrocytes, Journal of Orthopaedic Science (2016), http://dx.doi.org/10.1016/j.jos.2016.05.009
2
Y. Tang et al. / Journal of Orthopaedic Science xxx (2016) 1e7
directly, and it is not clear how to maintain a high concentration of TGF-b1 in cultured cells. Exogenous genes have been efficiently transferred into intervertebral disc cells using adenoviral vectors. The advantages of adenovirus-mediated gene delivery are that it is not influenced by the cell cycle, a large number of cells can accept multiple copies of recombinant adenovirus DNA, and transferred genes show prolonged expression during in vitro culture [11e13]. Therefore, in the present study, we attempted to transfer TGF-b1 using an adenoviral vector to achieve high concentrations in the matrix, and to investigate the effects of Adv-hTGF-b1 on chondrocyte function and ECM components. We established a recombinant hTGF-b1 overexpression system, and detected the effects of TGF-b1 on the regulation of type II collagen and aggrecan in a monolayer culture system and a 3D calciumealginate gel matrix culture system. This study assessed the application of TGF-b1 for the biological repair of cartilage. 2. Materials and methods
2.4. Calciumealginate gel bead culture A total of 2 106 chondrocytes were suspended in 1 ml 1.2% sodium alginate solution, then mixed with 102 mmol/L CaCl2 solution for 15 min without shaking, to form calciumealginate gel beads. Excess liquid was removed, and gel beads were washed three times using normal saline and DMEM medium. Ten beads were placed in each well of a 24-well tissue culture plate (Falcon) and cultured in 2 ml Ham's F12 medium supplemented with 10% FBS for 10 days. 2.5. Cell morphological analysis After culture in monolayers and 3D calciumealginate gel matrix culture systems for 10 days, the morphology of chondrocytes with or without Adv-hTGF-b1 transduction was examined by staining with the hematoxylin and eosin (HE) kit (Solarbio) or Alcian Blue kit (Shanghai yuanye Bio-Technology Co., Ltd) according to the manufacturer's instructions.
2.1. Chondrocyte isolation and culture
2.6. Quantitative fluorometric DNA analysis
The knee cartilage of New Zealand white rabbits was obtained from the tibial platform and the femoral condyle, in accordance with protocols approved by the Animal Care Committee of the Southern Medical University. Cartilage pieces (approximately 1 mm3) were treated with trypsin and type II collagenase (Sigma) overnight. Cells were then cultured in Ham's F12 medium supplemented with 10% fetal bovine serum (FBS, GIBCO-BRL) at 37 C with 5% CO2. The culture medium was refreshed every 2 days. The third generation of chondrocytes was used for the following study.
DNA content was evaluated by the bisbenzimidazole fluorescent dye assay. Briefly, 10 days after transfection, chondrocytes were digested in papain solution (125 mg/ml papain in phosphatebuffered saline, 5 mmol/L Na2EDTA, pH 6.0) and then reacted with 0.1 mg/ml bisbenzimidazole fluorescent dye in 10 mmol/L TriseHCl, pH 7.4. The DNA concentration was measured by a microplate reader with an excitation of 365 nm and an emission of 400e550 nm. 2.7. Quantitative RT-PCR
2.2. Recombinant adenoviral vector construction An adenoviral vector encoding TGFb1 (Adv-TGFb1) and a control null adenovirus (Adv-LacZ) were constructed as previously described [14,15]. First, Adv-TGFb1 mRNA was isolated from MCF10A cells, then cloned into a shuttle plasmid and cotransfected into human embryonic kidney 293 cells (ATCC, Rockville, MD) to generate the adenoviral vector. Adenoviral products were purified by CsCl gradient centrifugation and PD-10 Sephadex chromatog Que , Canada), then raphy (Amersham Pharmacia, Baie d'Urfe titered by plaque assays on the same 293 cells. Purified Adv-TGFb1 and Adv-LacZ adenoviruses were transfected into rabbit articular chondrocytes (see below), at which point the TGFb1 fragment was expressed as LAP-TGFb1. LAP was then cleaved off by protease furin in the Golgi apparatus, and finally excreted out of the cell [16]. All experimental protocols followed the Biosafety Committee of McMaster University (AUP-060313). 10 days later, supernatants were harvested and analyzed by enzyme-linked immunosorbent assays (ELISA; R&D Systems, Minneapolis, Min., USA) confirming TGFb1 overexpression. 2.3. Adv-hTGF-b1-transduced culture Chondrocytes were transfected with 200 multiplicity of infection of Adv-hTGF-b1 for 8 h, then the medium was changed to fresh Ham's F12 medium supplemented with 10% FBS. Primary untransduced chondrocytes and Lac-Z-transduced chondrocytes served as controls. Supernatants were harvested 10 days later and analyzed by enzyme-linked immunosorbent assays (R&D Systems, Minneapolis, MN) to detect the expression of TGFb1 and relevant ECM proteins.
After being cultured in monolayers and 3D calciumealginate gel matrix culture systems for 10 days, chondrocytes underwent total RNA extraction using the TriPure Isolation Reagent (Invitrogen Corp.). RNA was quantified by ultraviolet spectroscopy (Thermo Scientific), then 1 mg was reverse-transcribed into cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science). PCR amplification was performed using a Light Cycler Instrument and the Light Cycler Fast Start DNA Master SYBR Green 1 Kit (Roche Applied Science) according to the manufacturer's instructions. Finally, gene expression was quantified relatively by ABI Prism 7700 Sequence Detection System as a fold-increase compared with primary chondrocytes. Primer sequences are listed in Table 1. 2.8. Western blotting analysis Protein expression of TGF-b1, aggrecan, collagen-I, and collagenII were evaluated by western blotting after chondrocytes had been cultured for 10 and 36 days. Briefly, proteins were extracted from harvested chondrocytes and then 20 mL of protein was separated on 10% sodium dodecyl sulfate-polyacrylamide gels before being transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% dried milk at room temperature for 60 min. They were then incubated at 4 C overnight on a shaking platform with primary antibodies anti-b-actin (rabbit monoclonal antibody, SigmaeAldrich), anti-aggrecan (rabbit polyclonal antibody, SigmaeAldrich), anti-Col I (mouse monoclonal antibody, SigmaeAldrich), and anti-Col II (mouse monoclonal antibody, Invitrogen Corp.). Signals were detected using the enhanced chemiluminescence kit (Perkin Elmer, Waltham, MA) and quantified by the computer program ‘Quantity one’ (Bio-Rad).
Please cite this article in press as: Tang Y, et al., Effect of adenovirus-mediated TGF-b1 gene transfer on the function of rabbit articular chondrocytes, Journal of Orthopaedic Science (2016), http://dx.doi.org/10.1016/j.jos.2016.05.009
Y. Tang et al. / Journal of Orthopaedic Science xxx (2016) 1e7
3
Table 1 Primer sequences used for RT-qPCR. Gene
Forward
Reverse
SOX9 COL10A1 MMP13 b-actin
50 -ACCTCAAGAAGGAGAGCGAAG-30 50 -GGCTTCCCAGTGGCTGATAG-30 50 -GAATTAAGGAGCATGGCG AC-30 50 -AGACCACCTTCAACTCGATCAT-30
50 -CACCAGCGTCCAGTCGTAG-30 50 -TTTTGCTCTCTCTGGGTGGC-30 50 -TAAGGAGTGGCCGAACTCAT-30 50 -ACTCGTCATACTCCTGCTTGCT-30
2.9. Measurement of GAG contents After being cultured for 10 days, chondrocytes were treated using cell lysis buffer mixed with Alcian Blue solution (pH 2.0) to evaluate chronogenesis. They were washed with 40% dimethyl sulfoxide and 0.05 mol/L MgCl2, and dissolved in 4.0 mol/L guanidineeHCl and 33% propanol. The optical density of the extracted dye was measured on a 96-well plate reader at 600 nm.
several generations demonstrated long spindle-shaped fibrocytelike adherent growth. Adv-TGF-b1-transduced chondrocytes (T) showed a similar morphology to primary chondrocytes. We observed similar results following chondrocyte staining with HE and Alcian Blue. Chondrocytes cultured with 3D calciumealginate gel beads maintained their morphology, and almost no morphological changes were found among the four groups. 3.2. Analysis of TGF-b1 expression
2.10. Statistical analysis All data are presented as means ± SD and analyzed with SPSS 13.0 software. Comparisons were processed by one-way analysis of variance with p < 0.05 considered significant. 3. Results
Fig. 2A shows TGF-b1 expression levels in cells cultured for different times. TGF-b1 expression gradually increased over time up to the point at which chondrocytes had been cultured for 27 days, after which it declined. As shown in Fig. 2B, C, TGF-b1 expression was maintained at relatively low levels in Pri, NC, and BC groups, but was markedly increased in Adv-hTGF-b1-transduced chondrocytes in both culture systems after several generations.
3.1. Phenotypic analysis of chondrocytes in each culture system 3.3. Overexpression of TGF-b1 promotes DNA synthesis The morphology of chondrocytes cultured in monolayers and the 3D culture system is shown in Fig. 1. Primary chondrocytes (Pri) were shown to be round or oval, while Adv-LacZ transduced chondrocytes (NC) and blank control (BC) groups cultured for
The chondrocyte DNA content was determined by quantitative fluorometric analysis. In the monolayer culture system, TGF-b1 was found to promote DNA synthesis in chondrocytes compared with
Fig. 1. Morphological changes of chondrocytes cultured in both culture systems for 10 days were examined under a light microscope. Chondrocyte monolayers were stained with hematoxylin and eosin (HE) and Alcian Blue. Cells from 3D culture were captured using bright-field imaging without staining. Pri: primary chondrocytes; T: Adv-TGF-b1-transduced chondrocytes; NC: Adv-LacZ-transduced chondrocytes; BC: blank control (magnification, 100).
Please cite this article in press as: Tang Y, et al., Effect of adenovirus-mediated TGF-b1 gene transfer on the function of rabbit articular chondrocytes, Journal of Orthopaedic Science (2016), http://dx.doi.org/10.1016/j.jos.2016.05.009
4
Y. Tang et al. / Journal of Orthopaedic Science xxx (2016) 1e7
Fig. 2. Western blotting analysis of TGF-b1 expression in chondrocytes cultured for up to 36 days (A), for 10 days (B), and densitometric analysis (C). Data are mean ± SD of three experiments, *p < 0.05.
3.4. Effects of TGF-b1 on aggrecan and GAG levels Fig. 4 shows the absorbance value of GAG in chondrocyte supernatants. We found that this value was markedly higher in AdvhTGF-b1-transduced chondrocytes than in Adv-LacZ-transduced chondrocytes in both the monolayer and 3D culture systems. Moreover, western blot analysis revealed a similar trend of aggrecan expression with GAG synthesis, as shown in Fig. 5. A significant correlation was detected between the two indices, while aggrecan and GAG expression levels were relatively higher in the 3D culture system compared with the monolayer culture system.
Fig. 3. DNA content in rabbit chondrocytes cultured for 10 days as determined by quantitative DNA fluorometric analysis. Each data point represents the mean ± SD from three independent experiments, *p < 0.05.
LacZ transduction (Fig. 3). No significant difference was observed between Adv-LacZ-transduced chondrocytes and the BC group or between Adv-hTGF-b1-transduced chondrocytes and primary chondrocytes. In the 3D culture system, no significant difference was observed among all groups, which was consistent with the cellular morphology findings.
Fig. 4. Sulfated glycosaminoglycan (GAG) content in chondrocyte supernatants after being cultured for 10 days. Each data point represents the mean ± SD from three independent experiments, *p < 0.05.
3.5. Gene expression of chondrocytes RT-qPCR was used to assess gene expression of chondrocytes cultured in monolayers and the 3D culture system. As shown in Fig. 6, SOX9 mRNA expression was significantly decreased in NC and BC groups in both culture systems, but almost recovered to primary chondrocyte levels after Adv-hTGF-b1 transduction.
Fig. 5. Effect of gene delivery on aggrecan (Agn) expression in chondrocytes cultured for 10 days. (A) Agn protein levels were analyzed by western blot. (B) Densitometric analysis of Agn. Data points represent means ± SD from three independent experiments, *p < 0.05.
Please cite this article in press as: Tang Y, et al., Effect of adenovirus-mediated TGF-b1 gene transfer on the function of rabbit articular chondrocytes, Journal of Orthopaedic Science (2016), http://dx.doi.org/10.1016/j.jos.2016.05.009
Y. Tang et al. / Journal of Orthopaedic Science xxx (2016) 1e7
5
Fig. 6. RT-qPCR analysis of chondrocytes cultured in monolayer and 3D culture systems for 10 days. The induction of gene expression was determined relative to primary chondrocytes. Each data point represents the mean ± SD from three independent experiments, *p < 0.05.
Expression of the hypertrophic/mineralization markers COL10A1 and matrix metalloproteinase-13 (MMP13) were increased in NC and BC groups, but there were no significant differences between the T group and Pri group. These findings suggest that Adv-hTGF-b1 transduction promotes SOX9 mRNA expression and inhibits the expression of COL10A1 and MMP13 in monolayers and 3D culture systems.
3.6. Western blot analysis of type I and II collagen As shown in Fig. 7, western blot analysis revealed that primary chondrocytes mainly produced type II collagen (Fig. 7A, B) and low levels of type I collagen (Fig. 7A, C). The levels of type II collagen reduced and those of type I collagen increased after several generations in both culture systems. However, the trends of collagen expression reversed after Adv-hTGF-b1 transfection (Fig. 7B, C). Additionally, the ratio of expression between type II and type I collagen approached that seen in primary chondrocytes, especially in the 3D culture system (Fig. 7D). Taken together, these observations suggest that TGF-b1 maintained type II collagen expression in chondrocytes.
4. Discussion Previous cures for articular cartilage degeneration have mainly focused on autologous chondrocyte implantation, tissueengineering product replacement, and ex vivo gene therapy [17,18]. However, many issues still exist regarding these methods; for example, chondrocytes lose their original phenotype when cultured in a monolayer culture system. The delivery of an exogenous gene and the regulation of target cell function are also
difficult. One solution may be to overexpress a growth factor in autologous cells through genetic regulation. The reduction of chondrocyte activity and changes to cartilage ECM components always occur during subculture, so an appropriate chondrocyte phenotype is very important for its clinical application [19]. Within articular cartilage, the ECM plays a crucial role in regulating chondrocyte functions and metabolism. High levels of aggrecan and type II collagen are primary components of ECM [20]. In the present study, we found that primary chondrocytes cultured in vitro produce high levels of aggrecan and type II collagen, which decreases significantly after several generations of culture. The original primary chondrocyte phenotype also changed and its activity degraded after several generations in vitro. TGF-b1, as a member of the TGF-b superfamily, is a critical regulator of the development and differentiation of bone tissue. It regulates the production of collagen and aggrecan, and promotes bone marrow stromal cell (BMSC) proliferation and differentiation [21]. Tougu Xiaotong formula was reported to induce chondrogenic differentiation of BMSCs in association with TGF-b1 by activating the Ihh signaling pathway [22], while TGF-b1 expression in genetically modified human chondrocytes was proven to sustain positive effects on functional outcomes and cartilage growth in initial trials [23]. These studies demonstrate that TGF-b1 plays an important role in the development and maintenance of articular cartilage [24]. In the current study, we found that the expression of exogenous TGF-b1 was maintained after chondrocyte culture in monolayers and 3D culture systems for more than 1 month, with maximum levels reached after culture for 27 days. In the absence of exogenous TGF-b1, chondrocyte proliferation slowed down, and the cells resembled fibroblasts and eventually underwent apoptosis after in vitro culture. However, TGF-b1 overexpression through
Please cite this article in press as: Tang Y, et al., Effect of adenovirus-mediated TGF-b1 gene transfer on the function of rabbit articular chondrocytes, Journal of Orthopaedic Science (2016), http://dx.doi.org/10.1016/j.jos.2016.05.009
6
Y. Tang et al. / Journal of Orthopaedic Science xxx (2016) 1e7
Fig. 7. Col-I and col-II protein expression in chondrocytes cultured for 10 days. (A) col-I and col-II protein levels. Densitometric analysis of col-II (B) and col-I (C). (D) Ratio of col-II/ col-I protein expression. Data are mean ± SD of three experiments, *p < 0.05.
adenoviral transduction prevented the degradation of rabbit articular chondrocytes. TGF-b1 not only significantly promoted cell division and DNA synthesis, but also enhanced the synthesis of GAG and type II collagen. SOX9 is a chondrogenic transcription factor involved in chondrocyte differentiation and the expression of chondrocyte-specific anabolic genes such as type II collagen and aggrecan [25e27]. COL10A1 and MMP-13 have been widely interpreted as markers of hypertrophic or “osteoarthritic” chondrocytes, with COL10A1 an indicator of endochondral ossification, and MMP13 found to interfere with ECM synthesis [28,29]. In the present study, we found that expression of SOX9 was enhanced and that of COL10A1 and MMP-13 was inhibited after Adv-hTGF-b1 transduction of chondrocytes. Therefore, Adv-hTGF-b1 was beneficial in maintaining the phenotype of chondrocytes. The 3D calciumealginate culture system, as one of the best in vitro culture models, has been demonstrated to provide an environment close to the in vivo conditions necessary for assessing the functional status of nucleus pulposus cells [30]. To eliminate the influence of ECM components and tissue microenvironments in our study, chondrocytes were cultured within alginate gel beads in all groups. Although Adv-hTGF-b1-transduced chondrocytes expressed high levels of TGF-b1, DNA synthesis did not increase in cells grown in the 3D culture, indicating that there was no significant difference in cell proliferation between the TGF-b1-transduced group and control groups. However, the expression levels of aggrecan and type II collagen increased significantly after TGF-b1 transfection. The biological effect of TGF-b1 on chondrocytes may relate to certain temporary factors. Because the third generation of chondrocytes cultured in the 3D calciumealginate culture system
showed a similar phenotype to primary chondrocytes, this demonstrates that TGF-b1 released by gels gradually accumulated in Adv-hTGF-b1-transduced chondrocytes, then stimulated ECM synthesis and regulated the balance of ECM components. Thus, Adv-hTGF-b1-transduced chondrocytes appear to be a good prospect for re-implantation into degenerative joints. Moreover, the increased expression of ECM proteins regulated by TGF-b1 would be likely to recover the original environments surrounding articular chondrocytes and reverse articular degeneration. Furthermore, Adv-hTGF-b1-transduced articular chondrocytes should achieve a stable high concentration of TGF-b1 with no side-effects to adjacent tissue after transplantation. In conclusion, the overexpression of TGF-b1 was shown to be beneficial in improving the phenotype and function of chondrocytes. Therefore, TGF-b1 represents a potential therapeutic target for the modulation of cartilage development. However, further studies should be performed to investigate mechanisms of selective TGF-b1 targeting. Conflicts of interest All authors have seen and agree with the content of the manuscript and there is no conflict of interest to the report. References [1] Espanha MM. Articular cartilage: structure and histochemical composition. Acta Reumatol Port 2010 OcteDec;35(5):424e33. [2] Maldonado M, Nam J. The role of changes in extracellular matrix of cartilage in the presence of inflammation on the pathology of osteoarthritis. Biomed Res Int 2013;2013:284873.
Please cite this article in press as: Tang Y, et al., Effect of adenovirus-mediated TGF-b1 gene transfer on the function of rabbit articular chondrocytes, Journal of Orthopaedic Science (2016), http://dx.doi.org/10.1016/j.jos.2016.05.009
Y. Tang et al. / Journal of Orthopaedic Science xxx (2016) 1e7 [3] Hunziker EB, Lippuner K, Shintani N. How best to preserve and reveal the structural intricacies of cartilaginous tissue. Matrix Biol 2014 Oct;39:33e43. [4] Kim IL, Mauck RL, Burdick JA. Hydrogel design for cartilage tissue engineering: a case study with hyaluronic acid. Biomaterials 2011 Dec;32(34):8771e82. [5] Schagemann JC, Mrosek EH, Landers R, Kurz H, Erggelet C. Morphology and function of ovine articular cartilage chondrocytes in 3-D hydrogel culture. Cells Tissues Organs 2006 Jun;182(2):89e97. [6] Xu HG, Hu CJ, Wang H, Liu P, Yang XM, Zhang Y, Wang LT. Effects of mechanical strain on ANK, ENPP1 and TGF-beta1 expression in rat endplate chondrocytes in vitro. Mol Med Rep 2011 SepeOct;4(5):831e5. [7] Goldberg AJ, Lee DA, Bader DL, Bentley G. Autologous chondrocyte implantation. Culture in a TGF-beta-containing medium enhances the re-expression of a chondrocytic phenotype in passaged human chondrocytes in pellet culture. J Bone Jt Surg Br 2005 Jan;87(1):128e34. [8] Chen S, Emery SE, Pei M. Coculture of synovium-derived stem cells and nucleus pulposus cells in serum-free defined medium with supplementation of transforming growth factor-beta1: a potential application of tissue-specific stem cells in disc regeneration. Spine (Phila Pa 1976) 2009 May;34(12): 1272e80. [9] Liu H, Ning B, Wang D, Hu Y. Biological effects of recombinant adenoassociated virus 2 mediated human transforming growth factor beta1, encoding gene transfer to rabbit degenerative nucleus pulposus cells on proteoglycan level. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi 2010 May;24(5):618e21. [10] Li X, Hou X, Tang T. Increased synthesis of extracellular matrix in passaged nucleus pulposus cells by transfection with adenoviral vectors containing human transforming growth factor beta1. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi 2007 Dec;21(12):1342e7. [11] Zacchigna S, Zentilin L, Giacca M. Adeno-associated virus vectors as therapeutic and investigational tools in the cardiovascular system. Circulation Res 2014 May;114(11):1827e46. [12] Wold WSM, Toth K. Adenovirus vectors for gene therapy, vaccination and cancer gene therapy. Curr Gene Ther 2013 Dec;13(6):421e33. [13] Crystal RG. Adenovirus: the first effective in vivo gene delivery vector. Hum Gene Ther 2014 Jan;25(1):3e11. [14] Sime PJ, Xing Z, Graham FL, Csaky KG, Gauldie J. Adenovector-mediated gene transfer of active transforming growth factor-beta1 induces prolonged severe fibrosis in rat lung. J Clin Investig 1997 Aug;100(4):768e76. [15] Bett AJ, Haddara W, Prevec L, Graham FL. An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3. Proc Natl Acad Sci U S A 1994 Sep;91(19):8802e6. [16] Tritschler I, Gramatzki D, Capper D, Mittelbronn M, Meyermann R, Saharinen J, Wick W, Keski-Oja J, Weller M. Modulation of TGF-beta activity by latent TGF-beta-binding protein 1 in human malignant glioma cells. Int J Cancer 2009 Aug 1;125(3):530e40. [17] Makris EA, Gomoll AH, Malizos KN, Hu JC, Athanasiou KA. Repair and tissue engineering techniques for articular cartilage. Nat Rev Rheumatol 2015 Jan;11(1):21e34.
7
[18] Musumeci G, Castrogiovanni P, Leonardi R, Trovato FM, Szychlinska MA, Di Giunta A, Loreto C, Castorina S. New perspectives for articular cartilage repair treatment through tissue engineering: a contemporary review. World J Orthop 2014 Apr;5(2):80e8. [19] Ito A, Aoyama T, Iijima H, Tajino J, Nagai M, Yamaguchi S, Zhang X, Kuroki H. Culture temperature affects redifferentiation and cartilaginous extracellular matrix formation in dedifferentiated human chondrocytes. J Orthop Res 2015 May;33(5):633e9. [20] Gao Y, Liu S, Huang J, Guo W, Chen J, Zhang L, Zhao B, Peng J, Wang A, Wang Y, Xu W, Lu S, Yuan M, Guo Q. The ECMecell interaction of cartilage extracellular matrix on chondrocytes. Biomed Res Int 2014;2014:648459. [21] Miura Y, Parvizi J, Fitzsimmons JS, O'Driscoll SW. Brief exposure to high-dose transforming growth factor-beta1 enhances periosteal chondrogenesis in vitro: a preliminary report. J Bone Jt Surg Am 2002 May;84-A(5):793e9. [22] Chen J, Liu G, Weng X, Liu F, Lin P, Li H, Chen W, Huang Y, Liu X, Ye H, Li X. Tougu Xiaotong formula induces chondrogenic differentiation in association with transforming growth factor-beta1 and promotes proliferation in bone marrow stromal cells. Int J Mol Med 2015 Mar;35(3):747e54. [23] Elmallah RK, Cherian JJ, Jauregui JJ, Pierce TP, Beaver WB, Mont MA. Genetically modified chondrocytes expressing TGF-beta1: a revolutionary treatment for articular cartilage damage? Expert Opin Biol Ther 2015 Mar;15(3):455e64. [24] Huang X, Xu J, Huang M, Li J, Dai L, Dai K, Zhang X. Histone deacetylase1 promotes TGF-beta1-mediated early chondrogenesis through downregulating canonical Wnt signaling. Biochem Biophys Res Commun 2014 Oct;453(4):810e6. [25] de Crombrugghe B, Lefebvre V, Behringer RR, Bi W, Murakami S, Huang W. Transcriptional mechanisms of chondrocyte differentiation. Matrix Biol 2000 Sep;19(5):389e94. [26] Kulyk WM, Franklin JL, Hoffman LM. Sox9 expression during chondrogenesis in micromass cultures of embryonic limb mesenchyme. Exp Cell Res 2000 Mar;255(2):327e32. [27] Seifarth C, Csaki C, Shakibaei M. Anabolic actions of IGF-I and TGF-beta1 on interleukin-1beta-treated human articular chondrocytes: evaluation in two and three dimensional cultures. Histol Histopathol 2009 Oct;24(10):1245e62. [28] Caron MMJ, Emans PJ, Coolsen MME, Voss L, Surtel DAM, Cremers A, van Rhijn LW, Welting TJM. Redifferentiation of dedifferentiated human articular chondrocytes: comparison of 2D and 3D cultures. Osteoarthr Cartil 2012 Oct;20(10):1170e8. [29] Plaas A, Velasco J, Gorski DJ, Li J, Cole A, Christopherson K, Sandy JD. The relationship between fibrogenic TGFb1 signaling in the joint and cartilage degradation in post-injury osteoarthritis. Osteoarthr Cartil 2011 Sep;19(9): 1081e90. [30] Callejas FD, Martinez-Anton A, Alobid I, Fuentes M, Cortijo J, Picado C, RocaFerrer J, Mullol J. Reconstituted human upper airway epithelium as 3-D in vitro model for nasal polyposis. PLoS One 2014 Jun;9(6).
Please cite this article in press as: Tang Y, et al., Effect of adenovirus-mediated TGF-b1 gene transfer on the function of rabbit articular chondrocytes, Journal of Orthopaedic Science (2016), http://dx.doi.org/10.1016/j.jos.2016.05.009