pNNS-Conjugated Chitosan Mediated IGF-1 and miR-140 Overexpression in Articular Chondrocytes Improves Cartilage Repair

The aim of the present study was to investigate the effects of phosphorylatable nucleus localization signal linked nucleic kinase substrate short peptide (pNNS)-conjugated chitosan (pNNS-CS) mediated miR-140 and IGF-1 in both rabbit chondrocytes and cartilage defects model. pNNS-CS was combined with pBudCE4.1-IGF-1, pBudCE4.1-miR-140, and negative control pBudCE4.1 to form pDNA/pNNS-CS complexes. Then these complexes were transfected into chondrocytes or injected intra-articularly into the knee joints. High levels of IGF-1 and miR-140 expression were detected both in vitro and in vivo. Compared with pBudCE4.1 group, in vitro, the transgenic groups significantly promoted chondrocyte proliferation, increased glycosaminoglycan (GAG) synthesis, and ACAN, COL2A1, and TIMP-1 levels, and reduced the levels of nitric oxide (NO), MMP-13, and ADAMTS-5. In vivo, the exogenous genes enhanced COL2A1, ACAN, and TIMP-1 expression in cartilage and reduced cartilage Mankin score and the contents of NO, IL-1β, TNF-α, and GAG contents in synovial fluid of rabbits, MMP-13, ADAMTS-5, COL1A2, and COL10A1 levels in cartilage. Double gene combination showed better results than single gene. This study indicate that pNNS-CS is a better gene delivery vehicle in gene therapy for cartilage defects and that miR-140 combination IGF-1 transfection has better biologic effects on cartilage defects.


Introduction
Articular cartilage has limited self-repair ability [1]. Gene therapy is a good candidate for articular cartilage repair and has become a hot topic for research [1][2][3][4][5]. The choice of gene delivery vehicle is crucial to gene therapy, and many studies have been undertaken to develop efficient and safe gene delivery vehicles [2][3][4][5][6][7]. As a polycationic nonviral gene delivery vehicle, chitosan (CS) has been studied by many researchers. The effects of gene delivery of CS nanoparticles carrying therapeutic genes, microRNA (miRNA), or siRNA have been studied both in vitro and in vivo [6][7][8][9][10][11][12]. The transfection efficiency of CS is low under physiological conditions [9]. Many researchers, including us, have attempted to improve the transfection efficiency of CS through chemical modifications to its structure [6][7][8][9][10][11][12][13]. In our previous study, we have confirmed that p NNS-CS improved the pDNA transfection efficiency in C2C12 myoblast cells [13]. So we proposed that p NNS-CS can be used in the study of gene therapy for cartilage defects as a gene delivery vehicle. The structure of chondrocytes is different from that of C2C12 cell, and previously we only studied the transfection efficiency of p NNS-CS in vitro. So there are many problems need to be verified, such as how is the transfection efficiency of p NNS-CS in chondrocyte and in vivo, whether the intraarticular injection administration affects the stability of 2 BioMed Research International the pDNA/ p NNS-CS complex, and whether p NNS-CS is a reliable and efficient gene delivery vehicle in cartilage defects gene therapy. Therefore, the present study was designed to evaluate p NNS-CS as gene delivery vehicle carrying exogenous genes into chondrocyte both in vitro and in vivo.
The goals of this study were to determine whether p NNS-CS can carry IGF-1 and miR-140 genes into chondrocyte and efficient expression and the effects of exogenous genes both in cultured rabbit chondrocyte and in cartilage defects. The efficacies of the combination of IGF-1 and miR-140 have also been detected.  (Tokyo, Japan); ACAN, COL2A1, tissue inhibitor of  metalloproteinases-1 (TIMP1), matrix metallopeptidase-13  (MMP-13), and a disintegrin and metalloproteinase with thrombospondin motifs 5(ADAMTS-5) antibodies from Bioss (Beijing, China). One-week-old and three-month-old New Zealand white rabbits (2.0-2.5 kg) were purchased from Jinan Jinfeng Experimental Animal Limited by Share Ltd. (Shandong, China).

Isolation and Culture
Transfection of Articular Chondrocytes. Articular chondrocytes were isolated from knees of both hind limbs of one-week-old rabbits and cultured as described previously [8], and in the following experiments, the second-generation chondrocytes were used. Chondrocytes were seeded on 96-well and 6-well microplates in complete DMEM/F12 containing 10% FBS in an incubator containing 5% CO2 at 37 ∘ C. Chondrocytes were treated with pDNA/ p NNS-CS complexes when grown to 75% confluence. Chondrocytes were treated with IL-1 (10ng/mL) 24h after transfection.

Proliferation and Apoptosis of Chondrocytes.
Chondrocytes proliferation was detected using a standard MTT method. Cells were seeded in 96-well microplates and transfected with pDNA/ p NNS-CS complexes. MTT solution (15 L; 5 mg/mL) was applied to each well after 48 hours transfection and incubated at 37 ∘ C for another 4 hours. Then dimethylsulfoxide (150 L/well) was added to each well, and the optical density at 570 nm was detected, and background optical density at 630 nm was subtracted. Each group experiment was repeated six times.
Chondrocytes apoptosis was detected using Annexin V-FITC labeling by flow cytometry (BD, USA) as described previously [8].

NO, GAG, and IGF-1 Levels in Cell Supernatants.
Cell supernatants were collected after 96-hour transfection and detected to determine the accumulation of NO by a nitrate reductase kit, the concentrations of GAG, and IGF-1 by ELISA kits according to the manufacturer's directions.

Western Blot Analysis.
To detect the expression of collagen II, aggrecan, TIMP-1, MMP-13, and ADAMTS-5, chondrocytes transfection 96 h was lysed in RIPA lysis buffer (containing 0.1% PMSF). The concentration of protein was detected by bicinchoninic acid protein assay kit. The lysates were run on 8% SDS-polymerized gel and electrotransferred to PVDF membranes. The membranes were blocked in TBS-T containing 5% skimmed milk and incubated with primary antibody against collagen II , and GAPDH (1:500, using as the loading control), according to standard immunoblotting protocols. Proteins were detected using enhanced chemiluminescence western blot detection kit (Millipore, Darmstadt, Germany) according to the manufacturer's guide and pictures were captured using the Chemi-DocTM XRS+system (Bio-Rad, USA).

Animals and Experimental Articular Cartilage Defect.
Twenty-four three-month-old rabbits were randomly divided into four groups of six rabbits each. All four groups were made artificial cartilage full-thickness defects (4 mm diameter; 3 mm deep) as previously described [27] and received pDNA/pNNS-CS complexes. Following surgery, disinfection of the skin wounds and intramuscular injection of penicillin (400,000 U) were performed for 5 days. All rabbits were raised in separate cages under normal conditions, and allowed to exercise freely. Within 1 week after the operation, the joint activities of all rabbits in each group almost returned to normal. All procedures involving animals were approved by the Animal Care and Use Committee of China.
Eight weeks after surgery, all experimental rabbits were again anaesthetized and 1mL isotonic saline was injected to lavage the joint space. The joint cavity lavage fluid (synovial fluid) was used to detect the levels of NO, GAG, and IGF-1. Then all rabbits were killed, dissected, and photographed. The area of defect and its surrounding cartilage tissue were collected and divided into two parts. One part was used to extract total RNA for qRT-PCR (n=6), and another part was used to histological evaluation (n=6).

NO, IL-1 , TNF-, GAG, and IGF-1 Levels in Synovial
Fluids. The levels of NO, IL-1 , TNF-, GAG, and exogenous IGF-1 in synovial fluid were detected as described above in vitro study.

Histological Assay of the Articular Cartilage.
Following dissection, one-half of the cartilage defective area from each group was placed in the bottles containing 10% buffered formalin. After 24 h, the specimens were decalcified using 10% EDTA solution and embedded in paraffin. Serial sagittal sections were cut and stained with toluidine blue, Safranin O/fast green, and immunohistochemistry, following the standard operating procedure. Articular cartilage structure was observed using optical microscope (Olympus, Japan), and the severity of cartilage damage was graded histologically according to the Mankin scale [28].
2.6. Statistical Analysis. All results are reported as mean ± standard deviation (SD). Statistical significance was evaluated using SPSS 17.0 software. Multigroup comparisons were evaluated using the single-factor analysis of variance. Each result was compared using the Student-Newman-Keuls test. P <0.05 were considered to be statistically significant.

Agarose Gel Electrophoresis and Transfection
Efficiency of pDNA/ -CS Complexes. When the ratio of pDNA: p NNS-CS was at or beyond 1:2, the pDNA/ p NNS-CS complexes lost their mobility in the gel (Figure 1(a)). Fluorescent microscope showed that pEGFP-C1 was transfected into chondrocytes, and the p NNS conjugation increases the expression of EGFP gene (Figure 1(b)).

Discussion
CS can cross-link with collagen macromolecules [29]. Chondrocytes and collagen are exposed when cartilage is damaged, which facilitates the CS nanoparticles localization and exogenous genes expression in the defect, resulting in a therapeutic effect [30]. Our previous studies use p NNS (contained a potentially phosphorylatable serine residue and a SV40 nucleus localization signal) conjugated chitosan ( p NNS-CS) as gene delivery vehicle and in in vitro experiments have  confirmed that p NNS-CS could carry more pDNA into the nucleus of C2C12 cells and enhance exogenous genes expression, which is mainly related to the fact that p NNS can promote exogene nuclear localization and intranucleus disassociation [13]. The results drive us to use p NNS-CS as gene delivery vehicle in chondrocytes. Thus, the goal of this study is to further survey the effects of using p NNS-CS mediated gene transfection in chondrocyte, as well as the effects on cartilage defects repair. In this study, we first verified that p NNS-CS can improve transfection efficiency in chondrocytes. Second the results demonstrate that p NNS-CS mediated pBudCE4.1-IGF-1, pBudCE4.1-miR-140, or pBudCE4.1-IGF-1+miR-140 genes transfection in chondrocytes can induce IGF-1 and miR-140 overexpression both in vitro and in vivo.
Since IGF-1 promotes chondrocyte proliferation and ECM synthesis, it has been widely used in cartilage defect repair and has made encouraging results [4,8,15,16]. Many miRNAs play critical roles in cartilage-specific processes [31][32][33]. miR-140 inhibits the degradation of cartilage ECM by inhibiting ADAMTS5 and MMP13 expression [18,25,34,35]. Osteoarthritis and inflammatory signaling associated with cartilage degradation reduce miR-140 expression [34,36]. miR-140 −/− mice show early osteoarthritic changes onset in various cartilages, and miR-140 transgenic mice are resistant to arthritis induction [35,37]. Many studies have attempted to use miR-140 to interfere with cartilage-related diseases [18,25,26,37,38]. In view of the roles of IGF-1 and miR-140 in chondrocytes, we proposed that IGF-1 and miR-140 may have hopeful potential as therapeutic targets for cartilage defects treatment. Our experimental results show that introduction of IGF-1 and miR-140 by p NNS-CS transfection has a positive synergistic effect both in vitro and in vivo, and these effects of combination of two genes are obviously better than that of single gene. NO as an inflammatory mediator and catabolizing factor is closely related to the damage of cartilage, which can inhibit proteoglycan and COL2A1 synthesis and induce matrix metalloproteinase (MMP S ) synthesis in chondrocytes. High NO concentrations significantly induce chondrocyte apoptosis and decrease chondrocyte vitality [39,40], so inhibition of NO production is a potential strategy for the treatment of cartilage damage. In this study, the outcomes showed that overexpression of IGF-1 and miR-140 inhibits NO production, inhibits apoptosis, and promoted chondrocyte against of IL-1 antiproliferative effect, and IGF-1 and miR-140 jointly significantly enhance these effects both in vitro and in vivo. TNF-and IL-1 have been demonstrated to be important for cartilage degeneration. In this study, the outcomes showed that overexpression of IGF-1 and miR-140 reduces the content of TNF-and IL-1 in the synovial fluid, and IGF-1 and miR-140 jointly significantly enhance these effects in vivo.
ACAN, COL2A1, and GAG are known to be the most components of cartilage ECM. In vitro, ACAN, COL2A1, and GAG biosynthesis support chondrocyte redifferentiation [41]. Therefore, changes in ACAN, COL2A1, and GAG reflect the anabolism of cartilage ECM. In this study, in vitro, overexpression of IGF-1 and miR-140 each individually promoted GAG accumulation in the cell supernatant and chondrocyte expression of ACAN and COL2A1, and these effects were significantly enhanced in IGF-1 and miR-140 jointly group. In vivo, cartilage damage causes GAG to release into synovial fluids. So, the changes of GAG levels in synovial fluids reflect catabolic activity in cartilage ECM [42]. In this study, overexpression of IGF-1 and miR-140 each individually reduces the content of GAG in synovial fluid, decreases COL1A2 (fibrocartilaginous markers) and COL10A1 (cartilage hypertrophy markers) synthesis [43,44], and increases ACAN and COL2A1 synthesis compared with the negative control group (pBudCE4.1 transfection group) which were beneficial for cartilage repair, and fewer GAG in the synovial fluid, less COL1A2 and COL10A1, and more ACAN and COL2A1 synthesis were detected in IGF-1 combined with miR-140 group. These results imply that the repair tissue filling in the cartilage defects possesses characteristics of hyaline cartilage, and IGF-1 and miR-140 have synergistic effects for better therapeutic efficacy. ECM degrading enzymes, such as the matrix metalloproteinase-13 (MMP-13) and a metalloproteinase with thrombospondin Motifs-5 (ADAMTS-5), play important roles in cleaving ACAN and COL2A1 [6,45] and were involved in progressive erosion of articular cartilage. TIMP-1 is an inhibitor of MMPs activity during articular cartilage degeneration [46], which promotes cell proliferation and reduces cell apoptosis [18]. IGF-1 [16,45] and miR-140 [18,26,34,35,38] both can significantly reduce MMP-13 and ADAMTS-5 expression and increase TIMP-1 level, thus inhibiting the degeneration of ACAN and COL2A1 of ECM. In this study, IGF-1 and miR-140 each individually shows that beneficial effects on MMP-13, ADAMTS-5, and TIMP-1 are consistent with these results of previous studies [16,18,26,[34][35][36][37][38]47], and more beneficial effects were detected in IGF-1 and miR-140 jointly group; these may explain their mediated ECM production and chondrocyte proliferation.
Histological analysis also showed that intra-articular gene delivery of IGF-1 and miR-140 significantly lowered the Mankin score of defect cartilage, promoted ACAN, COL2A1, and GAG synthesis in the ECM, and diminished COL1A2 and COL10A1 staining intensities in the newly formed cartilage tissue. All results strongly suggest that the synergistic functions in promoting functional recovery of IGF-1 and miR-140 double transfection were obviously better than either of the single transfections, not only for inhibiting the inflammatory response, cartilage degradation, chondrocyte hypertrophy, and fibrous cartilage formation but also for promoting cartilage proliferation.

Conclusions
Our study verified that p NNS-CS can efficiently carry exogenous genes into chondrocytes and expression. Meanwhile, these study results provided the direct experimental evidences that gene therapy using IGF-1 and miR-140 was valid in repairing of cartilage defects, and combinations of IGF-1 and miR-140 have better biologic effects valid in improving repairing of articular cartilage and inhibiting degradation of articular cartilage. Our findings also provide a suitable experimental basis for articular cartilage defects gene therapy in vivo in the future.

Data Availability
The data used to support the findings of this study are included within the article.