TNFα and IL-1ß influence the differentiation and migration of murine MSCs independently of the NF-κB pathway.

INTRODUCTION: Mesenchymal stem cells (MSCs) have the ability to repair and regenerate tissue, home to sites of inflammation, and evade the host immune system. As such, they represent an attractive therapy for the treatment of autoimmune inflammatory diseases. However, results from in vivo murine studies in inflammatory arthritis have been conflicting, and this may be due to the genetic background of the MSCs used. It is known that the inflammatory milieu may influence properties of MSCs and that, in the case of human bone marrow-derived MSCs, this may be mediated by the nuclear factor-kappa-B (NF-κB) pathway. We sought to determine whether pro-inflammatory cytokines altered the differentiation and migration capacity of murine MSCs from different mouse strains and whether this was mediated by NF-κB. METHODS: The differentiation and migration of FVB and BALB/c MSCs were carried out in the presence of varying concentrations of tumor necrosis factor-alpha (TNFα) and interleukin (IL)-1ß, and the NF-κB pathway was inhibited in one of two ways: either by transduction of MSCs with an adenoviral vector expressing a super-repressor of NF-κB or by the addition of curcumin to culture media. RESULTS: Both BALB/c and FVB MSCs were sensitive to the effect of pro-inflammatory cytokines in vitro. TNFα and IL-1ß suppressed BALB/c osteogenesis and adipogenesis and FVB osteogenesis. The migration of both cell types toward media containing fetal bovine serum was augmented by pre-stimulation with either cytokine. In neither cell type were the cytokine effects reversed by abrogation of the NF-κB pathway. CONCLUSIONS: These data show that murine MSCs from different genetic backgrounds may be influenced by an inflammatory milieu in a manner that is not mediated by NF-κB, as is the case for human MSCs. This is not mediated by NF-κB. These findings are important and should influence how in vivo trials of murine MSCs are interpreted and the future development of pre-clinical studies in inflammatory diseases.

Facilitated endogenous repair: making tissue engineering simple, practical, and economical.

Facilitated endogenous repair is a novel approach to tissue engineering that avoids the ex vivo culture of autologous cells and the need for manufactured scaffolds, while minimizing the number and invasiveness of associated clinical procedures. The strategy relies on harnessing the intrinsic regenerative potential of endogenous tissues using molecular stimuli, such as gene transfer, to initiate reparative processes in situ. In the simplest example, direct percutaneous injection of an osteogenic vector is used to stimulate bone healing. If necessary, additional progenitor cells and space-filling scaffolds can be provided by autologous bone marrow, muscle, fat, and perhaps other tissues. These can be harvested, processed, and reimplanted by simple, expedited, intraoperative procedures. Examples of repair of experimental osseous and osteochondral lesions in laboratory animals are described. If successful, these strategies will provide methods for tissue regeneration that are not only effective but also inexpensive, safe, and clinically expeditious. Although orthopaedic examples are given here, the technology should be more generally applicable.

Dual transduction of insulin-like growth factor-I and interleukin-1 receptor antagonist protein controls cartilage degradation in an osteoarthritic culture model.

This study evaluated the potential of gene induced synoviocyte expression of a combination of insulin-like growth factor-I (AdIGF-I) and interleukin-1 receptor antagonist protein (AdIL-1Ra) to control articular cartilage degradation in vitro. Cartilage explants and synovial membrane were harvested from young mature horses. Synovial monolayers were established and either (1) maintained as untransduced controls; (2) transduced with AdIGF-I at 200 MOI in 500 microl serum-free medium; (3) transduced with AdIL-1Ra at 100 MOI; or (4) transduced with a combination of AdIGF-I (200 MOI) and AdIL-1Ra (100 MOI). Following transduction, cartilage explants were exposed to the synovial monolayer medium using co-culture inserts. Cultures were maintained for 6 days in either serum-free medium or medium containing 10 ng/ml recombinant human interleukin-1beta. At termination, synovial cell RNA was isolated for real-time PCR analysis, and cartilage explants were collected for H&E and toluidine blue staining, immunohistochemistry for type II collagen and IGF-I, in situ localization of IGF-I and type II collagen gene expression, and biochemical assays. Synovial monolayers were readily transduced with both AdIGF-I and AdIL-1Ra. IGF-I and IL-1Ra protein were secreted at beneficial levels throughout the experiment, having peak concentrations of 94.6 ng/ml and 33.0 ng/ml, respectively. Transduction with IGF-I promoted cartilage production of proteoglycan and type II collagen, suggesting a beneficial role for healing injured cartilage. Transduction with IL-1Ra decreased the synovial expression of IL-1alpha and IL-1beta and matrix metalloproteinases, indicating a mechanism for prevention of matrix degradation. The beneficial effects of the combination of anabolic growth factors and catabolic blockers were evident in improved preservation of proteoglycan content of cartilage explants exposed to the depleting effects of IL-1. These results show that gene therapy combining anabolic growth factors to stimulate matrix synthesis and catabolic blockers to prevent matrix degradation by IL-1, protects and causes partial restoration of cartilage matrix, and suggest a potential benefit of combination gene therapy for cartilage healing.

Evaluation of Ad-BMP-2 for enhancing fracture healing in an infected defect fracture rabbit model.

The objective of this study was to evaluate the use of adenoviral transfer of the BMP-2 gene (Ad-BMP-2) for enhancing healing in an infected defect fracture model. A femoral defect stabilized with plates and screws was surgically created in sixty-four skeletally mature New Zealand white rabbits. Experimental groups were: (1) non-infected Ad-luciferase (Ad-LUC, NONLUC), (2) non-infected Ad-BMP-2 (NONBMP), (3) infected Ad-LUC (INFLUC), and (4) infected Ad-BMP-2 (INFBMP). A sclerosing agent was applied to the ends of the bone at surgery to facilitate the development of osteomyelitis. Fracture healing was evaluated radiographically and histologically. Data were analyzed using an ANOVA, with statistical significance set as p<0.05. Rabbits in the non-infected and infected groups that were treated with Ad-BMP-2 had earlier initial- and bridging-callus formation, and a higher overall external callus grade compared to rabbits in the Ad-LUC groups. Rabbits in the Ad-LUC groups had more defect ossification compared to rabbits in the Ad-BMP-2 groups. There was a trend for rabbits in the Ad-BMP-2 group that were euthanized at 2 and 4 weeks after surgery to have more bone and cartilage compared to rabbits in the Ad-LUC group. The results of this study suggest that Ad-BMP-2 enhances the early stages of healing in an infected defect fracture. The results of our study were not as favorable as those reported in previous studies because animals healed by a large bridging callus and not by defect ossification. This could have been a result of the sclerosing agent, which may have damaged the cells in the defect.