Light-activated gene transduction of recombinant adeno-associated virus in human mesenchymal stem cells.

Deficiencies in skeletal tissue repair and regeneration lead to conditions like osteoarthritis, osteoporosis and degenerative disc disease. While no cure for these conditions is available, the use of human bone marrow derived-mesenchymal stem cells (HuMSCs) has been shown to have potential for cell-based therapy. Furthermore, recombinant adeno-associated viruses (rAAV) could be used together with HuMSCs for in vivo or ex vivo gene therapy. Unfortunately, the poor transduction efficiency of these cells remains a significant obstacle. Here, we describe the properties of ultraviolet (UV) light-activated gene transduction (LAGT) with rAAV in HuMSCs, an advance toward overcoming this limitation. Using direct fluorescent image analysis and real-time quantitative PCR to evaluate enhanced green fluorescent protein (eGFP) gene expression, we found that the optimal effects of LAGT with limited cytotoxicity occurred at a UV dose of 200 J/m(2). Furthermore, this UV irradiation had no effect on either the chondrogenic or osteogenic potential of HuMSCs. Significant effects of LAGT in HuMSCs could be detected as early as 12 h after exposure and persisted over 21 days, in a time and energy-dependent manner. This LAGT effect was maintained for more than 8 h after irradiation and required only a 10-min exposure to rAAV after UV irradiation. Finally, we show that the production of secreted TGFbeta1 protein from rAAV-TGFbeta1-IRES-eGFP infected to HuMSCs is highly inducible by UV irradiation. These results demonstrate that LAGT combined with rAAV is a promising procedure to facilitate gene induction in HuMSCs for human gene therapy.

Effect of anti-tumor necrosis factor-alpha gene therapy on wear debris-induced osteolysis.

BACKGROUND: Particle phagocytosis by macrophages induces the secretion of tumor necrosis factor-alpha, which is involved in the development of an osteolytic response. Therefore, we aimed to determine whether gene delivery of a soluble inhibitor of tumor necrosis factor-alpha (sTNFR:Fc) could prevent wear debris-induced osteolysis in a mouse model. sTNFR:Fc is a fusion protein containing the extracellular domain of the human type-I tumor necrosis factor receptor fused to the Fc region of mouse immunoglobulin. It acts by binding to tumor necrosis factor-alpha and preventing signaling through the membrane-bound tumor necrosis factor receptors. METHODS: An adenoviral vector encoding the LacZ gene (Ad.CMV-NlacZ) was propagated and was tested for its ability to transduce calvarial tissue. Ad.CMV-TNFR:Fc (encoding sTNFR:Fc) or Ad.CMV-NlacZ was administered to CBAxB6 mice in the presence or absence of titanium particles implanted onto the calvaria. Serum levels of sTNFR:Fc were measured with enzyme-linked immunosorbent assay, and the mice were killed on the tenth postoperative day for histological analysis. The experiments were repeated in athymic nude mice to avoid complications associated with the adenovirus-specific immune response. RESULTS: Administration of the control virus (Ad.CMV-NlacZ) transduced 10% of the cells in the periosteum. Ad.CMV-NlacZ treatment of sham-treated or titanium-treated animals induced significant bone resorption and osteoclastogenesis above control levels (that is, those in animals not treated with a virus). Treatment with the sTNFR:Fc virus did not reduce bone resorption or osteoclast numbers below control levels in CBAxB6 mice. In the athymic mice, no increase in the midline sagittal suture area or osteoclastogenesis was observed after treatment with the control vector and sTNFR:Fc gene therapy reduced the suture area to background levels. CONCLUSIONS: An immunologic response to Ad.CMV-NlacZ was most likely responsible for the increase in bone resorption and osteoclastogenesis in the animals treated with the control vector alone. In the athymic mice, in the absence of this immune response, sTNFR:Fc gene therapy reduced bone resorption in the midline sagittal suture area but had no effect on osteoclastogenesis.

Efficacy of etanercept for wear debris-induced osteolysis.

A major limitation of total joint arthroplasty is that up to 20% of patients require revision surgery to correct prosthetic loosening. Aseptic loosening is believed to result from the phagocytosis of wear debris particles by macrophages, which secrete proinflammatory cytokines that stimulate osteolysis. Tumor necrosis factor alpha (TNF-alpha) has been shown to be one of the prominent cytokines in this cascade and to be involved critically in the generation of particle-induced osteolysis. Etanercept is a soluble inhibitor of TNF-alpha, which is widely used for the treatment of rheumatoid arthritis. Here, we show this agent's ability to prevent wear debris-induced osteolysis. In vitro we show that Etanercept can inhibit directly osteoclastic bone resorption in a bone wafer pit assay, as well as cytokine production from titanium (Ti)-stimulated macrophages. Using a quantitative in vivo model of wear debris-induced osteolysis, we show that Etanercept prevents bone resorption and osteoclastogenesis. In mice treated with Etanercept at the time of osteolysis induction, bone resorption and osteoclast numbers were reduced to background levels in both normal and human TNF-alpha (hTNF-alpha) transgenic mice. In an effort to evaluate its effect on established osteolysis, Etanercept was administered 5 days after Ti implantation, and we observed that further osteolysis was prevented. These data support the concept that TNF-alpha is involved critically in osteoclastogenesis and bone resorption during periprosthetic osteolysis and suggest that soluble TNF-alpha inhibitors may be useful as therapeutic agents for the treatment of prosthetic loosening in humans.

Tumor necrosis factor-alpha/nuclear transcription factor-kappaB signaling in periprosthetic osteolysis.

Due to irreversible joint destruction caused by the various arthritides, more than 400,000 total joint arthroplasties are performed each year in the United States. As many as 20% of these require revision surgery because of aseptic loosening. The current paradigm to explain aseptic loosening is that wear debris generated from the prosthesis stimulates the release of proinflammatory cytokines (i.e., tumor necrosis factor-alpha and interleukins 1 and 6) following phagocytosis by resident macrophages. These cytokines, in turn, initiate an inflammatory response, with the development of an erosive pannus that stimulates bone resorption by osteoclasts. In support of this model, we have previously shown that human monocytes produce large quantities of tumor necrosis factor-alpha in response to titanium particles in vitro. In the current study, we characterized the role of tumor necrosis factor-alpha/nuclear transcription factor-kappaB signaling in the proinflammatory response to titanium particles in vitro and in vivo. Using the mouse macrophage cell line J774, we showed that these cells produce an amount of tumor necrosis factor-alpha in response to titanium particles similar to that produced by human peripheral blood monocytes. The production of tumor necrosis factor-alpha was preceded by a drop in cellular levels of inhibitory factor-kappaBalpha protein and translocation of p50/p65 nuclear transcription factor-KB to the nucleus 30 minutes after stimulation. Levels of tumor necrosis factor-alpha and inhibitory factor-kappaBalpha mRNA increased 30 minutes after stimulation, consistent with the activation of nuclear transcription factor-kappaB. Interleukin-6 mRNA was first seen 4 hours after the addition of the titanium particles, indicating that the production of this cytokine is secondary to the immediate nuclear transcription factor-kappaB response. To test the relevance of tumor necrosis factor-alpha/nuclear transcription factor-kappaB signaling in response to titanium particles in vivo, we adopted an animal model in which the particles were surgically implanted on the calvaria of mice. The animals displayed a dramatic histological response to the debris, with the formation of fibrous tissue and extensive bone resorption after only 1 week. With use of immunohistochemistry and tartrate-resistant acid phosphatase staining, tumor necrosis factor-alpha and osteoclasts were readily detected at the site of inflammation and bone resorption in the calvaria of the treated mice. By testing mice that genetically over-produce tumor necrosis factor-alpha (hTNFalpha-Tg), those defective in tumor necrosis factor-alpha signaling (TNF-RI-/-), and those that are nuclear transcription factor-kappaB1-deficient (NFkappaB1-/-), we evaluated the importance of tumor necrosis factor-alpha/nuclear transcription factor-kappaB signaling in the biological processes responsible for aseptic loosening. The hTNFalpha-Tg mice had a grossly exaggerated response, the TNF-RI(-/-) mice showed little evidence of inflammation or bone resorption, and the nuclear transcription factor-kappaB1(-/-) mice had an inflammatory response without bone resorption. On the basis of these results, we propose a model for periprosthetic osteolysis in which wear debris particles are phagocytosed by macrophages, resulting in the activation of nuclear transcription factor-kappaB and the production of tumor necrosis factor-alpha. Tumor necrosis factor-alpha directly induces fibroblast proliferation and tissue fibrosis and recruits or activates, or both, osteoclasts to resorb adjacent bone.

Quantitative small-animal surrogate to evaluate drug efficacy in preventing wear debris-induced osteolysis.

Individuals who suffer from severe joint destruction caused by the various arthritidies often undergo total joint arthroplasty. A major limitation of this treatment is the development of aseptic loosening of the prosthesis in as many as 20% of patients. The current paradigm to explain aseptic loosening proposes that wear debris generated from the prosthesis initiates a macrophage-mediated inflammatory response by resident macrophages, leading to osteoclast activation and bone resorption at the implant interface. No therapeutic interventions have been proved to prevent or inhibit aseptic loosening. The development of therapeutic strategies is limited due to the absence of a quantitative surrogate in which drugs can be screened rapidly in large numbers of animals. We have previously described a model in which titanium particles implanted on mouse calvaria induce an inflammatory response with osteolysis similar to that observed in clinical aseptic loosening. Here, we present new methods by which the osteolysis in this model can be quantified. We determined that 6-8-week-old mice in normal health have a sagittal suture area of 50 (+/-6) microm2, which contains approximately five osteoclasts. As a result of the titanium-induced inflammation and osteolysis, the sagittal suture area increases to 197 (+/-27) microm2, with approximately 30 osteoclasts, after 10 days of treatment. The sagittal suture area and the number of osteoclasts in the calvaria of sham-treated mice remained unchanged during the 10 days. We also determined the effects of pentoxifylline, a drug that blocks the responses of tumor necrosis factor-alpha to wear debris, and the osteoclast inhibitor alendronate. We found that both drugs effectively block wear debris-induced osteolysis but not osteoclastogenesis. In conclusion, we found the measurements made with this model to be reproducible and to permit quantitative analysis of agents that are to be screened for their potential to prevent aseptic loosening.