- Open Access
rAAV-mediated overexpression of TGF-β stably restructures human osteoarthritic articular cartilage in situ
- Jagadeesh K Venkatesan†1,
- Ana Rey-Rico†1,
- Gertrud Schmitt1,
- Anna Wezel1,
- Henning Madry1, 2 and
- Magali Cucchiarini1Email author
© Venkatesan et al.; licensee BioMed Central Ltd. 2013
- Received: 4 July 2013
- Accepted: 11 September 2013
- Published: 13 September 2013
Therapeutic gene transfer is of significant value to elaborate efficient, durable treatments against human osteoarthritis (OA), a slow, progressive, and irreversible disorder for which there is no cure to date.
Here, we directly applied a recombinant adeno-associated virus (rAAV) vector carrying a human transforming growth factor beta (TGF-β) gene sequence to primary human normal and OA chondrocytes in vitro and cartilage explants in situ to monitor the stability of transgene expression and the effects of the candidate pleiotropic factor upon the regenerative cellular activities over time.
Efficient, prolonged expression of TGF-β achieved via rAAV gene transfer enhanced both the proliferative, survival, and anabolic activities of cells over extended periods of time in all the systems evaluated (at least for 21 days in vitro and for up to 90 days in situ) compared with control (reporter) vector delivery, especially in situ where rAAV-hTGF-β allowed for a durable remodeling of OA cartilage. Notably, sustained rAAV production of TGF-β in OA cartilage advantageously reduced the expression of key OA-associated markers of chondrocyte hypertrophic and terminal differentiation (type-X collagen, MMP-13, PTHrP, β-catenin) while increasing that of protective TIMPs and of the TGF-β receptor I in a manner that restored a favorable ALK1/ALK5 balance. Of note, the levels of activities in TGF-β-treated OA cartilage were higher than those of normal cartilage, suggesting that further optimization of the candidate treatment (dose, duration, localization, presence of modulating co-factors) will most likely be necessary to reproduce an original cartilage surface in relevant models of experimental OA in vivo without triggering potentially adverse effects.
The present findings show the ability of rAAV-mediated TGF-β gene transfer to directly remodel human OA cartilage by activating the biological, reparative activities and by regulating hypertrophy and terminal differentiation in damaged chondrocytes as a potential treatment for OA or for other disorders of the cartilage that may require transplantation of engineered cells.
- Human osteoarthritis
- Articular cartilage
- rAAV gene transfer
Osteoarthritis (OA) is a major, widespread degenerative disease of the entire joint characterized by complex structural and functional tissue and cell alterations [1–5] for which there is no cure to date. OA has a multifactorial etiology, being influenced by both genetic, mechanical, and environmental factors [6–8]. The gradual and irreversible degradation of the articular cartilage in OA, associated with a remodeling of the subchondral bone and osteophyte formation, is the result of an impaired cartilage homeostasis (prevalence of catabolic events activated by biomechanical and pro-inflammatory mediators, failure of the chondrocytes to preserve and restore the metabolic balance) [9, 10]. Thus far, none of the pharmacological treatments and surgical options available to manage OA have allowed to reproduce the original cartilage integrity in patients. The design of new therapeutic approaches for OA is therefore of crucial importance to effectively and durably counteract the regular progression of the disease by activating regenerative processes in the chondrocytes as a means to re-equilibrate the disturbed cartilage balance.
Therapeutic gene transfer is a valuable tool to achieve this goal as it has the potential to allow for the production of factors over extended periods of time compared with the application of recombinant molecules with short pharmacological half-lives. While protection against cartilage breakdown was afforded by delivering sequences coding for agents with preventive and/or inhibitory activities (an IL-1 receptor antagonist - IL-1Ra, siRNAs against IL-1 or ADAMTS-5, soluble IL-1 and TNF receptors - sIL-1R and sTNFR, NF-κB inhibitors, kallistatin - KBP, thrombospontin-1 - TSP-1, Dickkopf-1 - DKK-1, pro-opiomelanocortin - POMC) [11–21], compensation for the loss of matrix elements and cells was not achieved to further re-establish an original cartilage surface in these various experimental systems. Instead, such effects have been ascribed, at least to some extent, to gene transfer of factors with anabolic and/or proliferative properties like proteoglycan 4 , the insulin-like growth factor I (IGF-I) [18, 23, 24], fibroblast growth factor 2 (FGF-2) [25, 26], bone morphogenetic proteins 2 and 4 (BMP-2, -4) [23, 27], and the transcription factor SOX9 [28, 29].
Yet, even in the presence of such agents, only partial cartilage resurfacing was noted, showing the need to identify other components of therapeutic value for improved gene transfer applications in OA. Equally important, the development of an effective treatment for OA will necessitate that the gene vehicle promotes the stable expression of a candidate sequence that can durably counteracts the slow and irreversible progression of the disease. In this regard, the transforming growth factor beta (TGF-β) is an attractive candidate owing to its prominent, pleiotropic effects upon cartilage formation, chondrocyte proliferation, and extracellular matrix (ECM) synthesis and to its ability to suppress IL-1-induced cartilage breakdown [30–33]. Yet, little is known on the effects of TGF-β gene transfer and overexpression in primary human OA articular chondrocytes and articular cartilage over relevant, extended periods of time. Most remarkably, Ulrich-Vinther et al.  reported that delivery of TGF-β via the promising recombinant adeno-associated virus (rAAV) vectors resulted in increased levels of type-II collagen and aggrecan and reduced expression of matrix metalloproteinase 3 (MMP-3) in human OA chondrocytes in vitro for about a week although effects at later time points were not documented. As a matter of fact, rAAV are among the most advantageous classes of vectors available for therapy to date, especially for use as a gene transfer system in OA. rAAV derived from a human non-pathogenic replication-defective virus carry no viral coding sequences in the recombinant genome, making them less immunogenic than adenoviral vectors [23, 35, 36]. rAAV can modify the quiescent chondrocytes both in vitro and in situ in their dense ECM at very high efficiencies and for prolonged periods of time, probably due to their small size (20 nm) and to a good maintenance of the constructs in the host under episomal forms [24, 26, 28, 34, 37, 38]. This is in marked contrast with nonviral  and adenoviral vectors [23, 35, 36] that mediate only short-term transgene expression, and with retroviral vectors [40, 41] that require cell division and selection and carry the risk of insertional mutagenesis following integration in the host genome.
In the present study, we tested whether efficient TGF-β overexpression can be achieved over prolonged periods of time via rAAV gene transfer in primary chondrocytes and explant cultures prepared from the articular cartilage of normal donors and OA patients (the ultimate targets for therapy), leading to enhanced levels of cell proliferation, survival, and matrix synthesis compared with control (reporter gene vector) treatment. We further analyzed the extent by which the candidate rAAV TGF-β treatment is capable of restructuring OA cartilage compared with normal (control) cartilage and explored the pathways potentially implicated in the remodeling processes.
All reagents were from Sigma (Munich, Germany) except for the dimethylmethylene blue (DMMB) dye (Serva, Heidelberg, Germany). The anti-TGF-β (V), anti-MMP-13 (72B-01), anti-TIMP-1 (C-20) and -TIMP-3 (W-18), anti-parathyroid hormone-related protein (PTHrP) (1D1), anti-β-catenin (E-5), and anti-TGF-β receptor I (activin receptor-like kinase-1 ALK1: C-20; ALK5: T-19) antibodies were from Santa Cruz Biotechnology (Heidelberg, Germany). The anti-type-II collagen (AF-5710) was antibody from Acris (Hiddenhausen, Germany). The anti-type-X collagen (COL-10) and anti-BrdU (BU-33) antibodies were from Sigma. Active TGF-β secretion was monitored with the hTGF-β Quantikine ELISA (DB100B; R&D Systems; Wiesbaden, Germany). The Cell Proliferation ELISA BrdU was from Roche Applied Science (Mannheim, Germany). The ApopTag® Plus Peroxidase In Situ Apoptosis Detection Kit was from Chemicon-Millipore (Schwalbach/Ts., Germany). The type-II collagen contents were measured with the native type-II collagen Arthrogen-CIA Capture ELISA kit (Chondrex, Redmond, WA, USA) and those for type-X collagen using a COL-10 ELISA (Antibodies-Online, Aachen, Germany).
Cartilage and cells
Human normal articular cartilage was obtained from unaffected knee joints removed during tumor surgery (n = 8, age 65–73). OA was excluded on safranin O-stained sections using the Mankin score  (score 1–2). OA cartilage was obtained from joints undergoing total knee arthroplasty (n = 14, age 65–78) (Mankin score 7–9). The study was approved by the Ethics Committee of the Saarland Physicians Council. Research has been performed in accordance with the Declaration of Helsinki involving human material. Informed consent has been obtained from all participants. Explant cultures and chondrocytes (passage 1–2) were prepared as previously described [24, 26, 28, 38].
Plasmids and rAAV vectors
rAAV-lacZ is an AAV-2-based plasmid [43, 44] carrying the lacZ gene encoding β-galactosidase under the control of the cytomegalovirus immediate-early (CMV-IE) promoter [24, 26, 28, 38]. rAAV-hTGF-β carries a 1.2-kb human transforming growth factor beta 1 (hTGF-β) cDNA fragment (intronless open reading frame from the ATG to the stop codon) (pORF9-hTGFB1) (Invivogen, Toulouse, France) that was cloned in rAAV-lacZ in place of lac Z (the fragment was confirmed by sequencing). rAAV were packaged as conventional (not self-complementary) vectors using a helper-free, two-plasmid transfection system in the 293 cell line (an adenovirus-transformed human embryonic kidney cell line) using the packaging plasmid pXX2 and the Adenovirus helper plasmid pXX6 as previously described . Vector preparations were purified by dialysis and titered by real-time PCR (about 1010 transgene copies/ml, with a ratio viral particles-to-functional vector of 500/1) [24, 26, 28, 38].
The vectors were applied to the samples based on concentrations previously tested [24, 26, 28]. Chondrocytes (2 × 104) were transduced with rAAV (40 μl, i.e. 8 × 105 functional recombinant viral particles; multiplicity of infection MOI = 40) and cultured for up to 21 days, while explant cultures were transduced by direct application of the vectors (40 μl) onto the surface of the samples and cultured for up to 90 days [24, 26, 28, 38].
Transgene (TGF-β) expression was monitored by indirect immunostaining using a specific antibody, a biotinylated secondary antibody (Vector Laboratories), and the ABC method (Vector Laboratories) using diaminobenzidine (DAB) as the chromogen. Samples were examined under light microscopy (Olympus BX 45; Hamburg, Germany) [24, 26, 28, 38]. Expression of TGF-β was also assayed by ELISA at the denoted time points (in vitro: days 5 and 21; in situ: days 21 and 90).
Histological and immunohistochemical analyses
Cell and explant cultures were fixed and explants were processed to stain paraffin-embedded sections (5 μm) using safranin O to detect proteoglycans and hematoxylin eosin (H&E) to detect cells [24, 26, 28]. Expression of type-II and type-X collagen, MMP-13, TIMP-1 and −3, PTHrP, β-catenin, and the TGF-β receptor I (ALK1 and ALK5) was detected with specific antibodies, biotinylated secondary antibodies, and the ABC method with DAB. Samples were examined under light microscopy (Olympus BX 45).
Cell proliferation and apoptosis assays
The proliferative activities were assessed by immunolabeling after BrdU incorporation . Briefly, BrdU was introduced at a final concentration of 3 μg/ml in the culture medium 24 h after rAAV transduction. Samples were immunochemically processed to monitor the proliferation rates with a specific anti-BrdU antibody, a biotinylated secondary antibody, and the ABC method with DAB. Proliferation was also assessed using the Cell Proliferation ELISA BrdU, with OD proportional to the cell numbers, as previously described . In situ, nuclear DNA fragmentation consistent with apoptosis was determined by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method [24, 26].
The transduction efficiencies (ratio of cells positive for TGF-β immunolabeling to the total number of cells on immunohistological sections), the cells positive for BrdU uptake (ratio of cells positive for BrdU immunolabeling to the total number of cells on immunohistological sections), the cell densities (cell numbers/mm2 of surface of the site evaluated on histological sections), the apoptotic cells (ratio of cells positive for TUNEL assay to the total number of cells on immunohistological sections), the safranin O staining intensities (ratio of tissue surface positively stained by safranin O to the total surface of the site evaluated on histological sections), the type-II or type-X collagen immunostaining intensities (ratio of tissue surface positively immunostained by type-II or type-X collagen to the total surface of the site evaluated on immunohistological sections), as well as the cells positive for the expression of MMP-13, TIMP-1 and −3, PTHrP, β-catenin, and the TGF-β receptor I (ALK1, ALK5, ALK1/ALK5 ratio) (ratio of cells positive for immunolabeling of each of these markers to the total number of cells on immunohistological sections) were measured at three random sites standardized for their surface or using ten serial histological and immunohistochemical sections for each parameter, test, and replicate condition to allow for calculation of standard deviations (SD). Analysis programs included SIS AnalySIS (Olympus), Adobe Photoshop (Adobe Systems, Unterschleissheim, Germany), and Scion Image (Scion Corporation, Frederick, MD, USA) [24, 26, 28].
Explant cultures were processed for the assays as previously described [24, 26, 28]. The DNA contents were determined using Hoechst 33258, the proteoglycan contents by binding to the DMMB dye, and those for type-II collagen and type-X collagen by ELISA [24, 26, 28, 47]. Data were normalized to total cellular proteins using a protein assay (Pierce Thermo Scientific, Fisher Scientific, Schwerte, Germany). All measurements were performed with a GENios spectrophotometer/fluorometer (Tecan, Crailsheim, Germany).
Each condition was performed in triplicate in three independent experiments with both types of cultures. Data were obtained by two individuals that were blinded with respect to the treatment groups. Values are expressed as mean ± standard deviation (SD). The t-test and Mann–Whitney Rank Sum Test were employed where appropriate. P values of less than 0.05 were considered statistically significant.
rAAV-mediated TGF-β overexpression in human normal and OA articular chondrocytes in vitro and in situ
The functionality of the rAAV-hTGF-β vector was first tested in human normal and OA primary chondrocyte cultures and articular cartilage explants.
Significant, durable (at least 90 days) TGF-β expression was also achieved in situ when applying rAAV-hTGF-β to cartilage explants compared with rAAV-lacZ (normal cartilage: from 724.5 ± 4.9 to 304.2 ± 2.2 versus 92.3 ± 1.1 to 55.2 ± 1.9 pg/ml/24 h between days 21 and 90; OA cartilage: from 987.7 ± 4.8 to 324.9 ± 4.3 versus 83.4 ± 2.1 to 58.1 ± 3.2 pg/ml/24 h between days 21 and 90; up to 11.8-fold difference, always P ≤ 0.001), with specific immunoreactivity observed both in the superficial and middle zones of the cartilage and showing again durable transduction efficiencies (up to 70%) (Figure 1B).
These results show that the current rAAV TGF-β vector is capable of modifying human normal and OA chondrocytes both in vitro and in situ, allowing for significant levels of transgene expression compared with control vector administration over extended periods of time, especially when the cells are embedded in their ECM (at least 90 days in situ).
Effects of rAAV-hTGF-β administration upon the cellular activities of human normal and OA articular chondrocytes in vitro and in situ
We next evaluated the ability of rAAV-mediated TGF-β overexpression to stimulate the proliferative and survival activities of chondrocytes in the systems tested above.
Further biochemical analyses in vitro next revealed significant and durable (from day 5 to day 21) increases in the proteoglycan and type-II collagen contents with TGF-β versus lacZ both in normal and OA cells (up to 11.5-fold difference, always P ≤ 0.001) (Figures 3C and D) while those for type-X collagen significantly and durably decreased (from day 5 to day 21) with TGF-β (up to 1.7-fold difference, P ≤ 0.001 in OA cells) (Figure 3E). Again, similar results were obtained in cartilage explant cultures in situ. An analysis of the proteoglycan and type-II collagen contents showed significant and durable (from day 21 to day 90) increases with TGF-β versus lacZ both in normal and OA cartilage (up to 8.2-fold difference, always P ≤ 0.001) (Figures 4E and G). These findings were substantiated by an analysis of the intensities of safranin O staining and of type-II collagen immunostaining (up to 17.4-fold difference, always P ≤ 0.001) (Figures 4F,H, 5A, and B). Again, these parameters were always higher with TGF-β in normal cartilage versus lacZ (always P ≤ 0.001). Also, the contents and immunostaining intensities for type-X collagen significantly and durably (from day 21 to day 90) decreased with TGF-β (up to 20.5-fold difference, P ≤ 0.001 in OA cartilage) (Figures 4I, J, and 5C).
These findings show that application of rAAV-hTGF-β is capable of both enhancing the proliferative and anabolic activities of human normal and OA chondrocytes in vitro and in situ while advantageously delaying their terminal differentiation. While the effects of TGF-β were in general more robust early on both in vitro and in situ (between 1.1- and 1.7-fold difference), probably due to higher levels of TGF-β expression over time (up to 3.04-fold difference), they remained significant vis-à-vis lacZ at the latest time points evaluated (always P ≤ 0.001).
Evaluation of the pathways allowing for the long-term protective effects of TGF-β via rAAV gene transfer in human normal and OA articular cartilage
To determine the mechanisms possibly involved in the processes of TGF-β-mediated cartilage remodeling over time via rAAV gene transfer, we investigated the expression of critical chondrocyte differentiation-related and OA-associated factors in the cartilage in situ at the latest time point evaluated in the study (90 days) among which MMP-13 (collagenase-3, a marker of terminal differentiation), the members of the protective TIMP family (TIMP-1 and −3), PTHrP (a hypertrophy-associated agent), β-catenin (a mediator of the Wnt signaling pathway associated with hypertrophy), and the TGF-β receptor I (protective ALK5 signaling pathway versus alternative opposing ALK1 route).
These findings indicate that treatment of human OA cartilage with the candidate rAAV TGF-β vector beneficially impacts the processes of chondrocyte hypertrophy and terminal differentiation in human OA chondrocytes in situ via the TGF-β signaling pathway.
Direct therapeutic gene transfer based on the use of the efficient and stable rAAV vectors is a promising tool to manage the irreversible progression of OA. In this regard, TGF-β might be a good candidate to achieve this goal due to its protective and reparative effects in the articular cartilage [32, 33]. Notably, Ulrich-Vinther et al.  reported that gene transfer of TGF-β via rAAV was capable of increasing the levels of key ECM components while decreasing those of MMP-3 over a one-week period of time in human OA chondrocytes in vitro, yet the benefits of such an approach upon the long-term remodeling of human OA cartilage especially in situ remain to be elucidated. In the present study, we therefore examined whether an rAAV-hTGF-β vector can effectively and durably modify primary human normal and OA articular chondrocytes in vitro and most importantly in cartilage explant cultures in situ, leading to a prolonged activation of remodeling activities compared with control treatment.
rAAV mediates successful overexpression TGF-β in human normal and OA articular chondrocytes in vitro and in situ
For the first time to our best knowledge, we show that efficient, sustained TGF-β expression can be promoted by rAAV gene transfer both in human normal and OA chondrocytes in vitro for at least 21 days and in human normal and OA cartilage explants in situ for at least 90 days, probably resulting from the persistence of rAAV in the targets , and with transduction efficiencies reaching 70-80% in these systems, in good agreement with previous findings using this class of vector [24, 26, 28, 34, 37, 38]. The levels of production achieved here early on in vitro with rAAV (up to 552.4 pg TGF-β/ml/24 h on day 5 at an MOI = 40) were in the range of those reported by Ulrich-Vinther et al. at a similar time point (5 ng/ml/24 h on day 8 at an MOI = 250) . For comparison, the levels of expression reached 60 ng/ml/24 h with a nonviral vector but in bovine chondrocytes and using a very high amount of plasmid (2 μg) , 2.5 ng/ml/24 h with an adenoviral vector at an MOI of 50 but in a human chondrocyte-like cell line , and 20–33 ng/105 cells/24 h (i.e. 4–7 ng/2 × 104 cells/24 h) in human chondrocytes with retroviral vectors but tested upon selection of transduced cells [40, 41]. However, only very short-term expression was noted with these classes of vectors (never beyond 4 days) while we describe an ongoing, significant synthesis until day 21 (up to 219.4 pg/ml/24 h). Most remarkably, and for the first time, we further evidenced a sustained production of TGF-β in situ via rAAV (up to 90 days), reaching levels of up to 987.7 pg/ml/24 h and occurring through the whole thickness of the cartilage, probably due to the ability of the small rAAV particles to penetrate the dense matrix [24, 26, 28, 38].
rAAV-mediated TGF-β overexpression activates the proliferative and anabolic activities of human normal and OA articular chondrocytes in vitro and in situ
The data further indicate that such high, maintained levels of rAAV-delivered TGF-β stimulated both the proliferative, survival, and biosynthetic activities of human normal and OA chondrocytes in vitro and in situ over time compared with control treatments, consistent with the properties of the growth factor [23, 34–36, 39–41]. A rigorous comparison of the effects of TGF-β resulting from rAAV gene transfer compared with other vector classes is difficult to establish as divergent assessment methods have been used in these earlier studies [23, 34–36, 39–41]. Nevertheless, it is noteworthy that only short-term effects of the growth factor have been demonstrated there (only some few days) or following cell selection, and mostly in in vitro settings, whereas we report prolonged effects both in vitro and most significantly in situ.
rAAV-mediated TGF-β overexpression delays chondrocyte hypertrophy and terminal differentiation in situ via the TGF-β signaling pathway
Furthermore, application of the current TGF-β construct led to advantageous decreases in the expression of key OA-associated markers of chondrocyte hypertrophic and terminal differentiation like type-X collagen, MMP-13, PTHrP, and β-catenin, again in agreement with the effects of this growth factor [49, 50]. In contrast, TGF-β overexpression increased (although to a lesser extent) the levels of protective TIMPs as previously described [51, 52], allowing nevertheless to beneficially influence the balance between TIMPs and MMP-13 and suggesting that other pathways might be implicated. Most strikingly, we show that efficient, sustained production of TGF-β via rAAV significantly enhanced the levels of the critical TGF-β receptor I as previously reported , both for the ALK1 and ALK5 signaling pathways but in a fashion that restored a favorable, original ALK1/ALK5 balance in OA cartilage like in control normal cartilage [54, 55], allowing to overcome the age- and OA-associated changes in TGF-β signaling  and probably resulting in the modulation of hypertrophic and terminal differentiation processes .
Interestingly, overexpression of TGF-β in the conditions applied here led to enhanced biological activities in human OA cells and cartilage compared with control normal cells and cartilage. It remains to be seen whether such prominent activities will not alter the cell activities and cartilage and joint integrity over time especially in vivo, in light of reports showing adverse effects of TGF-β delivery in experimental animal models (synovial inflammation and fibrosis, osteophyte formation) [57–62]. Still, in these studies, detrimental effects were evidenced when very high amounts of recombinant factor were applied (100–200 ng while we report up to 987.7 pg biologically active TGF-β/ml/24 h with rAAV in situ), in a dose-dependent and recurrent manner , or following adenoviral-mediated gene transfer at much higher doses than those used here (107-109versus 8 × 105 viral particles) [57–61]. It is also important to note that in all these studies, administration of the treatments was performed by intra-articular injection, a setting where the gene vector and recombinant factor can target all the tissues of the joint, allowing TGF-β to possibly exert chemoattractant, inflammatory, and chondrogenic effects especially upon the periosteum, subchondral bone, and synovium [63–65] that is highly permissive to gene transfer .
In any case, careful optimization of rAAV TGF-β delivery and expression in vivo (dose, duration, localization) will be necessary to establish an effective and appropriate treatment for human OA that takes advantage of the favorable actions of the growth factor over its potentially deleterious effects. Beside injecting low vector doses as performed here, the use of regulatable (tetracycline-sensitive), disease-inducible (NF-κB, COX-2, proinflammatory cytokines), or tissue-specific control elements (SOX9, type-II collagen, cartilage oligomeric matrix protein) may permit to modulate transgene expression compared with the strong CMV-IE promoter. Another important consideration will be to carefully decide on the route of administration. Instead of a conventional approach by intra-articular injection, direct local application of the vector preparation to the sites of cartilage injury might be more favorable to prevent dilution of the treatment in the joint space leading to undesirable dissemination and uptake by surrounding tissues. This will be practicable only when some cartilage surface is remaining like in early stages of OA and transplantation of TGF-β-modified cells might be needed for more advanced cases of the disease, having the further advantages of containing the TGF-β transgene  and avoiding transduction of other joint tissues. In this regard, it is interesting to note that Ha et al.  reported the feasibility of delivering retrovirally TGF-β-modified chondrocytes in patients with severe OA (TissueGene-C dose-escalating phase I clinical trial) with a trend toward efficacy and without serious adverse effects, in marked contrast with findings in experimental systems showing deleterious effects of TGF-β (inflammation, fibrosis, osteophyte formation) when provided at very high and repeated doses [57–62]. Again, rAAV might be best suited to develop such indirect, ex vivo trials as their high transduction efficiencies allow to use them without having to preselect the transduced cells compared with retroviral vectors [40, 41, 67].
Finally, administration of other candidates in conjunction with TGF-β (concomittently or sequentially) might be necessary, especially those that can specifically counteract the side effects of the growth factor or of its putative secondary mediators (fibrotic CTGF, BMP-2 in the case of osteophyte formation) like the inhibitory Smad6 and Smad7 and antagonist gremlin [58, 59, 69]. Alternatively, agents like IL-1Ra or IL-1 siRNA, sTNFR, NF-κB inhibitors, KBP, TSP-1, DKK-1, POMC, sFlt-1 (a VEGF antagonist) [11, 13–21, 27] might provide other good options to achieve this goal. Again rAAV might be a powerful tool to achieve these goals as combined gene transfer with this class of vector has been demonstrated in the current systems evaluated .
In summary, the results of the present study indicate that for the first time and in marked contrast with other classes of vectors, the direct, prolonged overexpression of TGF-β via rAAV vectors can efficiently stimulate the reparative activities of human normal and OA chondrocytes over time in vitro and most importantly in situ, contributing to the significant, proper remodeling of human OA cartilage. Future studies will allow to determine the benefits of applying the rAAV-hTGF-β construct in an appropriate, clinically relevant experimental OA model in vivo, requiring to translate first the current findings in the corresponding animal cells. The present findings validate the concept of using rAAV as an effective treatment for human OA.
OA is an incurable joint disease that disables millions of people worldwide, remaining very difficult to manage. Gene-based approaches may provide long-term treatments to restore an original structure and integrity in OA cartilage by rejuvenating resident (or transplanted) cells. The safe and highly efficient rAAV vectors are particularly well suited to treat OA that is not a life-threatening disease. Here, we showed the potency of an rAAV TGF-β vector to remodel human OA cartilage over extended, clinically relevant periods of time. The effects of this therapeutic vector in vivo and upon other affected tissues in the OA joint remain now to be investigated.
This work was supported by the German Research Society (Deutsche Forschungsgemeinschaft) (grants DFG CU 55/1-1,/1-2, and/1-3 to MC and HM) and the German Osteoarthritis Foundation (Deutsche Arthrose-Hilfe) (grants to MC and HM). The authors thank RJ Samulski (The Gene Therapy Center, University of North Carolina, Chapel Hill, NC, USA) and X Xiao (The Gene Therapy Center, University of Pittsburgh, Pittsburgh, PA, USA) for providing the genomic AAV-2 plasmid clones, the pXX2 and pXX6 plasmids, and the 293 cell line.
- Loeser RF, Goldring SR, Scanzello CR, Goldring MB: Osteoarthritis: a disease of the joint as an organ. Arthritis Rheum. 2012, 64: 1697-1707. 10.1002/art.34453.PubMed CentralView ArticlePubMedGoogle Scholar
- Lories RJ, Luyten FP: The bone-cartilage unit in osteoarthritis. Nat Rev Rheumatol. 2011, 7: 43-49. 10.1038/nrrheum.2010.197.View ArticlePubMedGoogle Scholar
- Lotz M: Osteoarthritis year 2011 in review: biology. Osteoarthritis Cartilage. 2012, 20: 192-196. 10.1016/j.joca.2011.11.015.PubMed CentralView ArticlePubMedGoogle Scholar
- Poole AR: Osteoarthritis as a whole joint disease. HSS J. 2012, 8: 4-6. 10.1007/s11420-011-9248-6.PubMed CentralView ArticlePubMedGoogle Scholar
- van den Berg WB: Osteoarthritis year 2010 in review: pathomechanisms. Osteoarthritis Cartilage. 2006, 19: 338-341.View ArticleGoogle Scholar
- Guilak F: Biomechanical factors in osteoarthritis. Best Pract Res Clin Rheumatol. 2011, 25: 815-823. 10.1016/j.berh.2011.11.013.PubMed CentralView ArticlePubMedGoogle Scholar
- Kraus VB, Burnett B, Coindreau J, Cottrell S, Eyre D, Gendreau M, Gardiner J, Garnero P, Hardin J, Henrotin Y, Heinegard D, Ko A, Lohmander LS, Matthews G, Menetski J, Moskowitz R, Persiani S, Poole AR, Rousseau JC, Todman M, OARSI FDA Osteooarthritis Biomarkers Working Group: Application of biomarkers in the development of drugs intended for the treatment of osteoarthritis. Osteoarthritis Cartilage. 2011, 19: 515-542. 10.1016/j.joca.2010.08.019.PubMed CentralView ArticlePubMedGoogle Scholar
- Sandell LJ: Etiology of osteoarthritis: genetics and synovial joint development. Nat Rev Rheumatol. 2012, 8: 77-89. 10.2174/157339712802083795.View ArticlePubMedGoogle Scholar
- Goldring MB, Otero M: Inflammation in osteoarthritis. Curr Opin Rheumatol. 2011, 23: 471-478. 10.1097/BOR.0b013e328349c2b1.PubMed CentralView ArticlePubMedGoogle Scholar
- Kapoor M, Martel-Pelletier J, Lajeunesse D, Pelletier JP, Fahmi H: Role of proinflammatory cytokines in the pathophysiology of osteoarthritis. Nat Rev Rheumatol. 2011, 7: 33-42. 10.1038/nrrheum.2010.196.View ArticlePubMedGoogle Scholar
- Chen LX, Lin L, Wang HJ, Wei XL, Fu X, Zhang JY, Yu CL: Suppression of early experimental osteoarthritis by in vivo delivery of the adenoviral vector-mediated NF-kappaBp65-specific siRNA. Osteoarthritis Cartilage. 2008, 16: 174-184. 10.1016/j.joca.2007.06.006.View ArticlePubMedGoogle Scholar
- Chu X, You H, Yuan X, Zhao W, Li W, Guo X: Protective effect of lentivirus-mediated siRNA targeting ADAMTS-5 on cartilage degradation in a rat model of osteoarthritis. Int J Mol Med. 2013, 31: 1222-1228.PubMedGoogle Scholar
- Fernandes J, Tardif G, Martel-Pelletier J, Lascau-Coman V, Dupuis M, Moldovan F, Sheppard M, Krishnan BR, Pelletier JP: In vivo transfer of interleukin-1 receptor antagonist gene in osteoarthritic rabbit knee joints: prevention of osteoarthritis progression. Am J Pathol. 1999, 154: 1159-1169. 10.1016/S0002-9440(10)65368-0.PubMed CentralView ArticlePubMedGoogle Scholar
- Frisbie DD, Ghivizzani SC, Robbins PD, Evans CH, McIlwraith CW: Treatment of experimental equine osteoarthritis by in vivo delivery of the equine interleukin-1 receptor antagonist gene. Gene Ther. 2002, 9: 12-20. 10.1038/sj.gt.3301608.View ArticlePubMedGoogle Scholar
- Ghivizzani SC, Lechman ER, Kang R, Tio C, Kolls J, Evans CH, Robbins PD: Direct adenovirus-mediated gene transfer of interleukin 1 and tumor necrosis factor alpha soluble receptors to rabbit knees with experimental arthritis has local and distal anti-arthritic effects. Proc Natl Acad Sci U S A. 1998, 95: 4613-4618. 10.1073/pnas.95.8.4613.PubMed CentralView ArticlePubMedGoogle Scholar
- Hsieh JL, Shen PC, Shiau AL, Jou IM, Lee CH, Teo ML, Wang CR, Chao J, Chao L, Wu CL: Adenovirus-mediated kallistatin gene transfer ameliorates disease progression in a rat model of osteoarthritis induced by anterior cruciate ligament transection. Hum Gene Ther. 2009, 20: 147-158. 10.1089/hum.2008.096.View ArticlePubMedGoogle Scholar
- Hsieh JL, Shen PC, Shiau AL, Jou IM, Lee CH, Wang CR, Teo ML, Wu CL: Intraarticular gene transfer of thrombospondin-1 suppresses the disease progression of experimental osteoarthritis. J Orthop Res. 2010, 28: 1300-1306. 10.1002/jor.21134.View ArticlePubMedGoogle Scholar
- Nixon AJ, Goodrich LR, Scimeca MS, Witte TH, Schnabel LV, Watts AE, Robbins PD: Gene therapy in musculoskeletal repair. Ann N Y Acad Sci. 2007, 1117: 310-327. 10.1196/annals.1402.065.View ArticlePubMedGoogle Scholar
- Oh H, Chun CH, Chun JS: Dkk-1 expression in chondrocytes inhibits experimental osteoarthritic cartilage destruction in mice. Arthritis Rheum. 2012, 64: 2568-2578. 10.1002/art.34481.View ArticlePubMedGoogle Scholar
- Santangelo KS, Nuovo GJ, Bertone AL: In vivo reduction or blockade of interleukin-1beta in primary osteoarthritis influences expression of mediators implicated in pathogenesis. Osteoarthritis Cartilage. 2012, 20: 1610-1618. 10.1016/j.joca.2012.08.011.PubMed CentralView ArticlePubMedGoogle Scholar
- Shen PC, Shiau AL, Jou IM, Lee CH, Tai MH, Juan HY, Lin PR, Liu GS, Wu CL, Hsieh HL: Inhibition of cartilage damage by pro-opiomelanocortin prohormone overexpression in a rat model of osteoarthritis. Exp Biol Med. 2011, 236: 334-340. 10.1258/ebm.2010.010319.View ArticleGoogle Scholar
- Ruan MZ, Erez A, Guse K, Dawson B, Bertin T, Chen Y, Jiang MM, Yustein J, Gannon F, Lee BH: Proteoglycan 4 expression protects against the development of osteoarthritis. Sci Transl Med. 2013, 5: 176ra134-View ArticleGoogle Scholar
- Smith P, Shuler FD, Georgescu HI, Ghivizzani SC, Johnstone B, Niyibizi C, Robbins PD, Evans CH: Genetic enhancement of matrix synthesis by articular chondrocytes: comparison of different growth factor genes in the presence and absence of interleukin-1. Arthritis Rheum. 2000, 43: 1156-1164. 10.1002/1529-0131(200005)43:5<1156::AID-ANR26>3.0.CO;2-M.View ArticlePubMedGoogle Scholar
- Weimer A, Madry H, Venkatesan JK, Schmitt G, Frisch J, Wezel A, Jung J, Kohn D, Terwilliger EF, Trippel SB, Cucchiarini M: Benefits of recombinant adeno-associated virus (rAAV)-mediated insulin-like growth factor I (IGF-I) overexpression for the long-term reconstruction of human osteoarthritic cartilage by modulation of the IGF-I axis. Mol Med. 2012, 18: 346-358.PubMed CentralView ArticlePubMedGoogle Scholar
- Chen B, Qin J, Wang H, Magdalou J, Chen L: Effects of adenovirus-mediated bFGF, IL-1Ra and IGF-1 gene transfer on human osteoarthritic chondrocytes and osteoarthritis in rabbits. Exp Mol Med. 2010, 42: 684-695. 10.3858/emm.2010.42.10.067.PubMed CentralView ArticlePubMedGoogle Scholar
- Cucchiarini M, Terwilliger EF, Kohn D, Madry H: Remodelling of human osteoarthritic cartilage by FGF-2, alone or combined with Sox9 via rAAV gene transfer. J Cell Mol Med. 2009, 13: 2476-2488. 10.1111/j.1582-4934.2008.00474.x.View ArticlePubMedGoogle Scholar
- Matsumoto T, Cooper GM, Gharaibeh B, Meszaros LB, Li G, Usas A, Fu FH, Huard J: Cartilage repair in a rat model of osteoarthritis through intraarticular transplantation of muscle-derived stem cells expressing bone morphogenetic protein 4 and soluble Flt-1. Arthritis Rheum. 2009, 60: 1390-1405. 10.1002/art.24443.View ArticlePubMedGoogle Scholar
- Cucchiarini M, Thurn T, Weimer A, Kohn D, Terwilliger EF, Madry H: Restoration of the extracellular matrix in human osteoarthritic articular cartilage by overexpression of the transcription factor SOX9. Arthritis Rheum. 2007, 56: 158-167. 10.1002/art.22299.View ArticlePubMedGoogle Scholar
- Li Y, Tew SR, Russell AM, Gonzalez KR, Hardingham TE, Hawkins RE: Transduction of passaged human articular chondrocytes with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9. Tissue Eng. 2004, 10: 575-584. 10.1089/107632704323061933.View ArticlePubMedGoogle Scholar
- O’Keefe RJ, Puzas JE, Brand JS, Rosier RN: Effect of transforming growth factor-beta on DNA synthesis by growth plate chondrocytes: modulation by factors present in serum. Calcif Tissue Int. 1988, 43: 352-358. 10.1007/BF02553278.View ArticlePubMedGoogle Scholar
- Redini F, Galera P, Mauviel A, Loyau G, Pujol JP: Transforming growth factor beta stimulates collagen and glycosaminoglycan biosynthesis in cultured rabbit articular chondrocytes. FEBS Lett. 1988, 234: 172-176. 10.1016/0014-5793(88)81327-9.View ArticlePubMedGoogle Scholar
- Malemud CJ: The role of growth factors in cartilage metabolism. Rheum Dis Clin North Am. 1993, 19: 569-580.PubMedGoogle Scholar
- Van Beuningen HM, van der Kraan PM, Arntz OJ, van den Berg WB: Protection from interleukin 1 induced destruction of articular cartilage by transforming growth factor beta: studies in anatomically intact cartilage in vitro and in vivo. Ann Rheum Dis. 1993, 52: 185-191. 10.1136/ard.52.3.185.PubMed CentralView ArticlePubMedGoogle Scholar
- Ulrich-Vinther M, Stengaard C, Schwarz EM, Goldring MB, Soballe K: Adeno-associated vector mediated gene transfer of transforming growth factor-beta1 to normal and osteoarthritic human chondrocytes stimulates cartilage anabolism. Eur Cell Mater. 2005, 10: 40-50.PubMedGoogle Scholar
- Arai Y, Kubo T, Kobayashi K, Takeshita K, Takahashi K, Ikeda T, Imanishi J, Takigawa M, Hirasawa Y: Adenovirus vector-mediated gene transduction to chondrocytes: in vitro evaluation of therapeutic efficacy of transforming growth factor-beta 1 and heat shock protein 70 gene transduction. J Rheumatol. 1997, 24: 1787-1795.PubMedGoogle Scholar
- Shuler FD, Georgescu HI, Niyibizi C, Studer RK, Mi Z, Johnstone B, Robbins PD, Evans CH: Increased matrix synthesis following adenoviral transfer of a transforming growth factor beta1 gene into articular chondrocytes. J Orthop Res. 2000, 18: 585-592. 10.1002/jor.1100180411.View ArticlePubMedGoogle Scholar
- Arai Y, Kubo T, Fushiki S, Mazda O, Nakai H, Iwaki Y, Imanishi J, Hirasawa Y: Gene delivery to human chondrocytes by an adeno-associated virus vector. J Rheumatol. 2000, 27: 979-982.PubMedGoogle Scholar
- Madry H, Cucchiarini M, Terwilliger EF, Trippel SB: Recombinant adeno-associated virus vectors efficiently and persistently transduce chondrocytes in normal and osteoarthritic human articular cartilage. Hum Gene Ther. 2003, 14: 393-402. 10.1089/104303403321208998.View ArticlePubMedGoogle Scholar
- Shi S, Mercer S, Eckert GJ, Trippel SB: Regulation of articular chondrocyte aggrecan and collagen gene expression by multiple growth factor gene transfer. J Orthop Res. 2012, 30: 1026-1031. 10.1002/jor.22036.PubMed CentralView ArticlePubMedGoogle Scholar
- Lee DK, Choi KB, Oh IS, Song SU, Hwang S, Lim CL, Hyun JP, Lee HY, Chi GF, Yi Y, Yip V, Kim J, Lee EB, Noh MJ, Lee KH: Continuous transforming growth factor beta1 secretion by cell-mediated gene therapy maintains chondrocyte redifferentiation. Tissue Eng. 2005, 11: 310-318. 10.1089/ten.2005.11.310.View ArticlePubMedGoogle Scholar
- Song SU, Cha YD, Han JU, Oh IS, Choi KB, Yi Y, Hyun JP, Lee HY, Chi GF, Lim CL, Ganjei JK, Noh MJ, Kim SJ, Lee DK, Lee KH: Hyaline cartilage regeneration using mixed human chondrocytes and transforming growth factor-beta1-producing chondrocytes. Tissue Eng. 2005, 11: 1516-1526. 10.1089/ten.2005.11.1516.View ArticlePubMedGoogle Scholar
- Mankin HJ, Dorfman H, Lippiello L, Zarins A: Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. II. Correlation of morphology with biochemical and metabolic data. J Bone Joint Surg Am. 1971, 53: 523-537.PubMedGoogle Scholar
- Samulski RJ, Chang LS, Shenk T: A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication. J Virol. 1987, 61: 3096-3101.PubMed CentralPubMedGoogle Scholar
- Samulski RJ, Chang LS, Shenk T: Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression. J Virol. 1989, 63: 3822-3828.PubMed CentralPubMedGoogle Scholar
- Xiao X, Li J, Samulski RJ: Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J Virol. 1998, 72: 2224-2232.PubMed CentralPubMedGoogle Scholar
- Cucchiarini M, Ekici M, Schetting S, Kohn D, Madry H: Metabolic activities and chondrogenic differentiation of human mesenchymal stem cells following recombinant adeno-associated virus-mediated gene transfer and overexpression of fibroblast growth factor 2. Tissue Eng Part A. 2011, 17: 1921-1933. 10.1089/ten.tea.2011.0018.View ArticlePubMedGoogle Scholar
- Venkatesan JK, Ekici M, Madry H, Schmitt G, Kohn D, Cucchiarini M: SOX9 gene transfer via safe, stable, replication-defective recombinant adeno-associated virus vectors as a novel, powerful tool to enhance the chondrogenic potential of human mesenchymal stem cells. Stem Cell Res Ther. 2012, 3: 22-36. 10.1186/scrt113.PubMed CentralView ArticlePubMedGoogle Scholar
- Shi S, Mercer S, Eckert GJ, Trippel SB: Growth factor regulation of growth factor production by multiple gene transfer to chondrocytes. Growth Factors. 2013, 31: 32-38. 10.3109/08977194.2012.750652.PubMed CentralView ArticlePubMedGoogle Scholar
- Dong Y, Drissi H, Chen M, Chen D, Zuscik MJ, Schwarz EM, O’Keefe RJ: Wnt-mediated regulation of chondrocyte maturation: modulation by TGF-beta. J Cell Biochem. 2005, 95: 1057-1068. 10.1002/jcb.20466.PubMed CentralView ArticlePubMedGoogle Scholar
- Tchetina EV, Antoniou J, Tanzer M, Zukor DJ, Poole AR: Transforming growth factor-beta2 suppresses collagen cleavage in cultured human osteoarthritic cartilage, reduces expression of genes associated with chondrocyte hypertrophy and degradation, and increases prostaglandin E(2) production. Am J Pathol. 2006, 168: 131-140. 10.2353/ajpath.2006.050369.PubMed CentralView ArticlePubMedGoogle Scholar
- Gunther M, Haubeck HD, van de Leur E, Blaser J, Bender S, Gutgemann I, Fischer DC, Tschesche H, Greiling H, Heinrich PC, Graeve L: Transforming growth factor beta 1 regulates tissue inhibitor of metalloproteinases-1 expression in differentiated human articular chondrocytes. Arthritis Rheum. 1994, 37: 395-405. 10.1002/art.1780370314.View ArticlePubMedGoogle Scholar
- Su S, Grover J, Roughley PJ, DiBattista JA, Martel-Pelletier J, Pelletier JP, Zafarullah M: Expression of the tissue inhibitor of metalloproteinases (TIMP) gene family in normal and osteoarthritic joints. Rheumatol Int. 1999, 18: 183-191. 10.1007/s002960050083.View ArticlePubMedGoogle Scholar
- Shlopov BV, Gumanovskaya ML, Hasty KA: Autocrine regulation of collagenase 3 (matrix metalloproteinase 13) during osteoarthritis. Arthritis Rheum. 2000, 43: 195-205. 10.1002/1529-0131(200001)43:1<195::AID-ANR24>3.0.CO;2-G.View ArticlePubMedGoogle Scholar
- Blaney Davidson EN, Remst DF, Vitters EL, Van Beuningen HM, Blom AB, Goumans MJ, van den Berg WB, van der Kraan P: Increase in ALK1/ALK5 ratio as a cause for elevated MMP-13 expression in osteoarthritis in humans and mice. J Immunol. 2009, 182: 7937-7945. 10.4049/jimmunol.0803991.View ArticlePubMedGoogle Scholar
- Finnson KW, Parker WL, Ten Dijke P, Thorikay M, Philip A: ALK1 opposes ALK5/Smad3 signaling and expression of extracellular matrix components in human chondrocytes. J Bone Miner Res. 2008, 23: 896-906. 10.1359/jbmr.080209.View ArticlePubMedGoogle Scholar
- van der Kraan PM, Goumans MJ, Blaney Davidson E, Ten Dijke P: Age-dependent alteration of TGF-beta signalling in osteoarthritis. Cell Tissue Res. 2012, 347: 257-265. 10.1007/s00441-011-1194-6.PubMed CentralView ArticlePubMedGoogle Scholar
- Bakker AC, van de Loo FA, Van Beuningen HM, Sime P, Van Lent PL, van der Kraan PM, Richards CD, van den Berg WB: Overexpression of active TGF-beta-1 in the murine knee joint: evidence for synovial-layer-dependent chondro-osteophyte formation. Osteoarthritis Cartilage. 2001, 9: 128-136. 10.1053/joca.2000.0368.View ArticlePubMedGoogle Scholar
- Blaney Davidson EN, Vitters EL, Van Beuningen HM, van de Loo FA, van den Berg WB, van der Kraan PM: Resemblance of osteophytes in experimental osteoarthritis to transforming growth factor beta-induced osteophytes: limited role of bone morphogenetic protein in early osteoarthritic osteophyte formation. Arthritis Rheum. 2007, 56: 4065-4073. 10.1002/art.23034.View ArticlePubMedGoogle Scholar
- Blaney Davidson EN, Vitters EL, van den Berg WB, van der Kraan PM: TGF beta-induced cartilage repair is maintained but fibrosis is blocked in the presence of Smad7. Arthritis Res Ther. 2006, 8: R65-R72. 10.1186/ar1931.PubMed CentralView ArticlePubMedGoogle Scholar
- Mi Z, Ghivizzani SC, Lechman E, Glorioso JC, Evans CH, Robbins PD: Adverse effects of adenovirus-mediated gene transfer of human transforming growth factor beta 1 into rabbit knees. Arthritis Res Ther. 2003, 5: R132-R139. 10.1186/ar745.PubMed CentralView ArticlePubMedGoogle Scholar
- Remst DF, Blaney Davidson EN, Vitters EL, Blom AB, Stoop R, Snabel JM, Bank RA, van den Berg WB, van der Kraan PM: Osteoarthritis-related fibrosis is associated with both elevated pyridinoline cross-link formation and lysyl hydroxylase 2b expression. Osteoarthritis Cartilage. 2013, 21: 157-164. 10.1016/j.joca.2012.10.002.View ArticlePubMedGoogle Scholar
- Van Beuningen HM, Glansbeek HL, van der Kraan PM, van den Berg WB: Differential effects of local application of BMP-2 or TGF-beta 1 on both articular cartilage composition and osteophyte formation. Osteoarthritis Cartilage. 1998, 6: 306-317. 10.1053/joca.1998.0129.View ArticlePubMedGoogle Scholar
- Miura Y, Fitzsimmons JS, Commisso CN, Gallay SH, O’Driscoll SW: Enhancement of periosteal chondrogenesis in vitro. Dose–response for transforming growth factor-beta 1 (TGF-beta 1). Clin Orthop Relat Res. 1994, 301: 271-280.PubMedGoogle Scholar
- Nishimura K, Solchaga LA, Caplan AI, Yoo JU, Goldberg VM, Johnstone B: Chondroprogenitor cells of synovial tissue. Arthritis Rheum. 1999, 42: 2631-2637. 10.1002/1529-0131(199912)42:12<2631::AID-ANR18>3.0.CO;2-H.View ArticlePubMedGoogle Scholar
- Zhen G, Wen C, Jia X, Li Y, Crane JL, Mears SC, Askin FB, Frassica FJ, Chang W, Yao J, Carrino JA, Cosgarea A, Artemov D, Chen Q, Zhao Z, Zhou X, Riley L, Sponseller P, Wan M, Lu WW, Cao X: Inhibition of TGF-beta signaling in mesenchymal stem cells of subchondral bone attenuates osteoarthritis. Nat Med. 2013, 19: 704-712. 10.1038/nm.3143.PubMed CentralView ArticlePubMedGoogle Scholar
- Nita I, Ghivizzani SC, Galea-Lauri J, Bandara G, Georgescu HI, Robbins PD, Evans CH: Direct gene delivery to synovium. An evaluation of potential vectors in vitro and in vivo. Arthritis Rheum. 1996, 39: 820-828. 10.1002/art.1780390515.View ArticlePubMedGoogle Scholar
- Noh MJ, Copeland RO, Yi Y, Choi KB, Meschter C, Hwang S, Lim CL, Yip V, Hyun JP, Lee HY, Lee KH: Pre-clinical studies of retrovirally transduced human chondrocytes expressing transforming growth factor-beta-1 (TG-C). Cytotherapy. 2010, 12: 384-393. 10.3109/14653240903470639.View ArticlePubMedGoogle Scholar
- Ha CW, Noh MJ, Choi KB, Lee KH: Initial phase I safety of retrovirally transduced human chondrocytes expressing transforming growth factor-beta-1 in degenerative arthritis patients. Cytotherapy. 2012, 14: 247-256. 10.3109/14653249.2011.629645.PubMed CentralView ArticlePubMedGoogle Scholar
- Scharstuhl A, Vitters EL, van der Kraan PM, van den Berg WB: Reduction of osteophyte formation and synovial thickening by adenoviral overexpression of transforming growth factor beta/bone morphogenetic protein inhibitors during experimental osteoarthritis. Arthritis Rheum. 2003, 48: 3442-3451. 10.1002/art.11328.View ArticlePubMedGoogle Scholar
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