Introduction
Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage breakdown, synovial fibrosis, and bone spurs. An imbalance between catabolic and anabolic factors favoring the catabolic side is very likely involved in the pathological features of OA.
Currently, many attempts are being made to repair the cartilage that has been damaged in OA. One approach focuses on shifting the metabolic imbalance back by stimulating the anabolic side. Transforming growth factor-β (TGF-β) is one of the anabolic factors involved in cartilage maintenance and appears to be a good candidate for cartilage repair. TGF-β is a stimulator of extracellular matrix production, like collagen type II and proteoglycan (PG), in chondrocytes and it downregulates matrix-degrading enzymes [
1]. High amounts of TGF-β are stored in healthy cartilage [
2‐
6], whereas in OA cartilage the expression of TGF-β is reduced [
7]. Injection of TGF-β into naive murine knee joints results in an increase in PG content of the articular cartilage [
8]. Moreover, in murine experimental rheumatoid arthritis, injection of TGF-β protected cartilage from PG loss [
9]. In addition, TGF-β counteracts the anabolic factor interleukin-1 (IL-1), which is a very potent inducer of cartilage degradation [
10,
11] both
in vivo and
in vitro [
1,
12‐
16]. These data indicate that TGF-β has great potential as a tool for stimulating cartilage repair.
To obtain sufficient amounts of TGF-β in the joint for a prolonged period of time, an adenovirus can be used as a vehicle.
In vitro, chondrocytes that are transfected with an adenovirus encoding TGF-β responded by elevation of PG and collagen production [
17]. We wanted to assess whether adenoviral overexpression of TGF-β in the synovial lining could stimulate repair of damaged cartilage
in vivo.
Unfortunately, introducing high amounts of TGF-β into a knee joint has adverse effects. Administration of 20 ng TGF-β is already sufficient to result in an increased cellularity of the synovial lining, expansion of fibroblast population in the synovial connective tissue, and continued collagen deposition [
18]. Injection of high amounts of TGF-β, either as a bolus injection or via adenoviral transfection, results in marked hyperplasia of the synovium and chondro-osteophyte formation [
8,
18‐
21]. This illustrates that the use of TGF-β for cartilage repair can result in side effects that are deleterious for future therapeutic applications.
The aim of this study was to use TGF-β as a cartilage repair factor but at the same time to prevent the TGF-β-induced fibrotic side effect. Therefore, we examined the effect of adenoviral overexpression of active TGF-β on cartilage repair and additionally studied whether simultaneous Smad7 overexpression could block TGF-β-induced fibrosis. Smad7 is an intracellular molecule that inhibits the TGF-β signaling pathway. TGF-β binds to its type II receptor, which then forms a complex with the type I TGF-β receptor.
Subsequently, the intercellular signaling molecule Smad2 or Smad3 gets phosphorylated, forms a complex with common Smad, Smad4, and shuttles to the nucleus for transcription [
22]. Smad7 inhibits Smad2 and Smad3 phosphorylation, thereby preventing further signaling [
23,
24].
To both stimulate cartilage and block side effects, we took advantage of the fact that adenoviruses, once injected into the murine knee joint, transfect the synovial lining but do not penetrate the cartilage [
25]. We co-transfected the synovial lining with an adenovirus overexpressing TGF-β and an adenovirus overexpressing Smad7. The transfected synovial lining cells will produce TGF-β but due to an intercellular signaling block caused by Smad7, will no longer respond to this factor.
We show that adenoviral overexpression of TGF-β results in increased PG content of the cartilage both after IL-1-induced damage and in a spontaneous model of experimental OA. In both cases, the TGF-β-induced fibrosis can be prevented by simultaneous Smad7 overexpression.
Materials and methods
Animals
C57Bl/6 mice (10 weeks old) and STR/ort mice (4 weeks old) were used. Mice were kept in filter-top cages with woodchip bedding under standard pathogen-free conditions. They were fed a standard diet and tap water ad libitum. The local animal committee had approved this study.
Stimulation of cartilage repair by TGF-β after IL-1 insult
To assess whether adenoviral overexpression of TGF-β could stimulate cartilage repair, we inflicted cartilage damage in 73 C57Bl/6 mice by intra-articular injection of 10 ng IL-1 (R&D Systems, Inc., Minneapolis, MN, USA). Two days after IL-1 injection, PG synthesis will have reached a low point [
11]. At this time point, an adenovirus overexpressing active TGF-β (Ad-TGF-β
223/225) was injected intra-articularly (plaque-forming units [pfu] 10
7/6 μl) and compared with a control virus (Ad-del 70-3). Four days after the primary insult, 53 mice were used for patellae isolation for PG synthesis measurement by
35SO
42- incorporation. The other 20 mice were used for isolation of whole knee joints for histology.
Blocking TGF-β-induced fibrosis
To block TGF-β-induced fibrosis, 24 C57Bl/6 mice were injected intra-articularly with adenoviruses in the combinations of Ad-TGF-β223/225 + Ad-luciferase (Ad-luc), Ad-Smad7 + Ad-luc, and Ad-TGF-β223/225 + Ad-Smad7 (at a pfu of 0.5 × 107 per adenovirus in 6 μl) or Ad-luc alone (at a total pfu of 107) as a control. After 14 days, when synovial fibrosis can be observed histologically, knee joints were isolated for histology.
Simultaneously stimulating cartilage repair and blocking of fibrosis
To make sure that Smad7 did not interfere with TGF-β-stimulated PG synthesis, we assessed whether stimulation of cartilage repair was not blocked by co-transfection with Ad-Smad7, and cartilage damage was again introduced in 48 C57Bl/6 mice by intra-articular injection with 10 ng IL-1. After 2 days, mice were injected with adenoviruses in the combinations of Ad-TGF-β223/225 + Ad-luc, Ad-TGF-β223/225 + Ad-smad7, or Ad-luc alone. Four days after IL-1 injection, 24 mice were used for isolation of patellae for 35SO42- incorporation measurements. After 2 weeks, the other mice were used for isolation of knee joints for histological assessment of fibrosis.
Cartilage repair while blocking fibrosis in spontaneous OA
To test whether we could stimulate cartilage repair in a spontaneous experimental OA model while preventing fibrosis, we extended our experiment to STR/ort mice. STR/ort mice develop OA spontaneously and show pathological changes by 8 weeks of age. We injected adenoviruses intra-articularly into the knee joint of 24 4-week-old STR/ort mice and repeated this injection after 2 weeks. The adenoviruses were injected in the combinations of Ad-TGF-β223/225 + Ad-luc, Ad-smad7 + Ad-luc, and Ad-TGF-β223/225 + Ad-smad7 at a pfu of 0.5 × 107 per adenovirus or Ad-luc at a pfu of 107 alone as a control. Four weeks after the first injection, knee joints were isolated for histological analysis of synovial fibrosis and PG content of the cartilage.
Histology
Knee joints of mice were dissected and fixed in phosphate-buffered formalin for 7 days. Thereafter, they were decalcified in 10% formic acid for 1 week. Knee joints were dehydrated with an automated tissue-processing apparatus (Tissue Tek VIP, Sakura, Ramsey, MN, USA) and embedded in paraffin. Coronal whole knee joint sections of 7 μm were made. Sections were stained with Safranin O and Fast Green.
Immunohistochemistry
Sections were deparaffinized and washed with phosphate-buffered saline (PBS). For antigen unmasking, sections were incubated in citrate buffer (0.1 M sodium citrate + 0.1 M citric acid) for 2 hours. Endogenous peroxidase was blocked with 1% hydrogen peroxide in methanol for 30 minutes. Thereafter, sections were blocked with 5% normal serum of the species in which the secondary antibody was produced. Specific primary antibodies against procollagen type I (2 μg/ml) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were incubated overnight at 4°C. After extensive washing with PBS, the appropriate biotin-labeled secondary antibody was used (DakoCytomation, Glostrup, Denmark) for 30 minutes at room temperature followed by a biotin-streptavidine detection system according to manufacturer's protocol (Vector Laboratories, Burlingame, CA, USA). Bound complexes were visualized using DAB (3,3'-diaminobenzidine) reagent, counterstained with haematoxylin, dehydrated, and mounted with Permount.
PG synthesis
For measurement of PG synthesis,
35SO
42- was incorporated into isolated patellae. Immediately after isolation, the patellae were placed in Dulbecco's modified Eagle's medium with gentamicin (50 mg/ml) and pyruvate. After half an hour, medium was replaced by medium containing
35SO
42- (20 μCi/ml) and incubated for 3 hours at 37°C in 5% CO
2. Patellae were then further prepared for measurement of
35SO
42- incorporation in the articular cartilage as previously described [
26].
PG content
PG content was measured in sections stained with Safranin O and Fast Green, using a computerized imaging system as previously described [
27]. Briefly, Safranin O stains PGs in the cartilage red. The amount of PGs is determined by a computerized calculation of the amount of blue light passing through the red-stained cartilage. An increase in PGs leads to more intense red staining and reduced blue light passing through. The PG content of the tibia was calculated by the average of three sections per joint.
Measurement of fibrosis
Sections were stained immunohistochemically for procollagen type I as a measure of fibrosis. Subsequently, the amount of cells that stain positive in the synovial tissue was determined. A blinded observer selected the synovial tissue in three sections per knee joint. A computerized imaging system subsequently determined the amount of positive cells in the selected area. The obtained values were averaged per knee joint.
In addition, synovial hyperplasia was assessed by measurement of synovial thickness. This was determined in sections stained with Safranin O and Fast Green. The thickness of the synovial tissue was measured with a computerized imaging system again in three sections per knee joint and averaged per joint as previously described [
27] (Qwin; Leica Imaging Systems Ltd., Cambridge, UK). In short, the width of the joint from bone edge to joint capsule, minus the width of the joint space itself, was determined.
Statistical analysis
Results were analyzed with a Student's t test and stated significant if the p value was lower than 0.05.
Discussion
The cartilage damage in OA is thought to be a consequence of a misbalance between anabolic and catabolic factors, favoring the catabolic side. In this study, we used TGF-β as the anabolic factor for cartilage repair. TGF-β has been reported to enhance periosteal chondrogenesis in explants in a dose-dependent manner [
28]. Morales and colleagues [
4,
5] demonstrated that TGF-β increased PG synthesis and suppressed its degradation in articular cartilage organ cultures. In addition, van Beuningen and colleagues [
8] showed that
in vivo TGF-β injections result in prolonged elevation of PG synthesis and PG content of cartilage in mice. These studies indicate that TGF-β has good potential for repairing cartilage. We showed that adenoviral overexpression of TGF-β was indeed able to boost cartilage repair
in vivo.
In vitro, it had already been shown that chondrocytes exposed simultaneously to IL-1 and TGF-β could reverse the IL-1-induced suppression of PG incorporation in their extracellular matrix [
15]. Supportive of our findings, van Beuningen and colleagues [
13] demonstrated that,
in vivo, TGF-β also counteracted deleterious effects of IL-1 on cartilage PG synthesis and PG content. In the current study, we first damaged cartilage by IL-1 injection and subsequently overexpressed TGF-β. In this way, we could assess whether TGF-β was able to restore, instead of prevent, cartilage damage. We introduced TGF-β via adenoviral overexpression, thereby gaining prolonged high expression of TGF-β, instead of via a bolus injection that results in short TGF-β exposure. This way, we were able to demonstrate increased PG synthesis and higher PG content in cartilage not only in a clean setting introducing cartilage damage with IL-1 but also in a spontaneous OA model.
The drawback of using TGF-β is that it can have adverse effects in joints. TGF-β is a known inducer of fibrosis in various tissues, and synovial tissue is no exception [
8,
21]. We took advantage of the fact that adenoviruses transfect only the synovial lining. In addition, we profited from the fact that Smad7 is an intercellular inhibitor of TGF-β. Smad7 stays inside the cell that is transfected with the adenovirus encoding Smad7. Because the synovial lining is where TGF-β induces synovial fibrosis, by co-transfection with Smad7, the lining appeared to be less sensitive to TGF-β-induced fibrosis. The reduction of TGF-β-induced fibrosis was not optimal and resulted in only a partial block of the fibrosis. This is likely due to the fact that not all cells in the synovial lining will be targeted. By optimizing this, we might be able to target every single one of the synovial lining cells and thereby fully block the TGF-β-induced fibrosis.
We have previously demonstrated that blocking TGF-β with Ad-Smad7 in OA resulted in reduction of the synovial fibrosis that was induced by the OA process itself [
27]. Now we combined the Smad7 adenovirus with Ad-TGF-β to block the TGF-β-induced fibrosis. We showed that the Ad-TGF-β transfection was still functional in combination with Smad7. Moreover, we demonstrated for the first time that adenoviral overexpression of TGF-β could stimulate repair of damaged cartilage and that co-expression with Smad7 could prevent a great deal of the TGF-β-induced synovial fibrosis. Combining Smad7 and TGF-β resulted in a higher PG synthesis after IL-1 insult than did TGF-β alone. This is likely due to the reduced synovial fibrosis when combined with Smad7.
Unfortunately, synovial fibrosis is not the only side effect of TGF-β overexpression in knee joints. TGF-β can induce osteophyte formation [
8,
19,
20,
29‐
31]. In the case of OA, TGF-β can aggravate the osteophyte formation that already occurs. We show that it is possible to target synovial cells to prevent fibrosis. In a similar fashion, we could potentially target the mesenchymal stem cells that eventually form the osteophytes after TGF-β exposure. This could be an option when key players of osteophyte formation are identified and can be blocked selectively.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
ENBD participated in the animal experiments and immunohistochemistry, carried out the histological measurements, analyzed the data, and drafted the manuscript. ELV participated in the animal experiments, carried out histological processing of the knee joints, participated in immunohistochemistry, and performed 35S-sulphate measurements. PMK conceived of the study, participated in the design and coordination, and helped draft the manuscript. WBB participated in study design and revision of the final manuscript. All authors read and approved the final manuscript.