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We have developed an ex vivo method for delivering genes to the synovial lining of joints and expressing them intra-articularly. The present studies were designed to determine whether transfer of a human interleukin-1 receptor antagonist protein (IRAP) gene by this method was able to antagonize the intra-articular actions of interleukin-1. Intra-articular injections of human recombinant interleukin-1 beta (hrIL-1 beta) into the knees of control rabbits provoked a marked leukocytic infiltrate into the joint space, severe synovial thickening and hypercellularity, and loss of proteoglycans from articular cartilage. Genetically modified knees contained several nanograms of human IRAP and inhibited each of these effects of IL-1 beta. These data demonstrate for the first time that delivery of an appropriate gene to joints can prevent intra-articular pathology. Such findings permit cautious optimism about the eventual development of a gene treatment for arthritis and other disorders of the joint.

Almost all cell types release extracellular vesicles (EVs), which are derived either from multivesicular bodies or from the plasma membrane. EVs contain a subset of proteins, lipids, and nucleic acids from the cell from which they are derived. EV factors, particularly small RNAs such as miRNAs, likely play important roles in cell-to-cell communication both locally and systemically. Most of the functions associated with EVs are in the regulation of immune responses to pathogens and cancer, as well as in regulating autoimmunity. This Review will focus on the different modes of immune regulation, both direct and indirect, by EVs. The therapeutic utility of EVs for the regulation of immune responses will also be discussed.

Human LIM mineralization protein (LMP)-3 is one of the three splice variants of LMP recently identified. LMPs are involved in the osteoblast differentiation program and structurally characterized by the two conserved LIM and PDZ domains. Human LMP-1 (hLMP-1) shows one N-terminal PDZ domain and three C-term LIM domains connected by a non-conserved Unique region, deleted in the hLMP-2. hLMP-3 misses almost completely the LIM domains along with part of the unique region, due to a frame shift mutation. The three isoforms are expressed almost ubiquitously but show quantitative differences, hLMP-3 being the less expressed in all the analyzed tissues. Both hLMP-1 and hLMP-3 has been demonstrated to induce bone formation in vitro and ectopic bone formation in vivo, while hLMP-2 is not osteoinductive, suggesting that LIM domains are not essential for this function. Thus it has been hypothesized that the osteoinductive domain could reside in the Unique region that is partially conserved in hLMP-3. To examine the osteoinductive properties of this minimal domain we have cloned three different length of the Unique region of the hLMP-3 gene, corresponding to 120, 90 and 60 bp, fused to the enhanced green fluorescent protein (eGFP) and named L40-eGFP, L30-eGFP and L20-eGFP respectively. Thus we tested the ability of these constructs to induce bone specific gene expression and bone mineralization in vitro and ectopic bone formation in vivo in comparison to the full-length gene hLMP-3. Here we demonstrate that adenoviral-mediated gene transfer of all the 3 domains induces expression of certain bone-specific genes in a mouse fibroblasts cell line. The up-regulation of osteo-specific genes was assessed in mouse fibroblasts also by means of biolistic transfection using a plasmid containing a L20-eGFP fusion gene. In addition, we demonstrate that all the domains are able to induce mineralization in fibroblast and mesenchymal stem cells. An experiment to evaluate if direct gene transfer of the three constructs into murine skeletal muscle results in ectopic bone formation as efficiently as using LMP-3 is being performed. Finally in order to propose these new constructs as an effective approach to induce bone formation in vivo for clinical applications, we have synthesized a peptide of 20 aminoacid, corresponding to the fragment of 60 bp of the Unique region (named PTD-OD-1). The peptide enter the cells by a protein transduction domain (PTD-5) and its ability to induce in vitro expression of bone-specific genes and bone mineralization both in fibroblast and in human mesenchymal stem cells will be evaluated. PTD-OD-1 could represent a safe and powerful tool for clinical applications, and merit several analysis to evaluate its ability.

Transplantation of adult stem cells is being used to facilitate repair or regeneration of damaged or diseased tissues. However, in many cases, the therapeutic effects of the injected stem cells are mediated by factors secreted by stem cells and not by differentiation of the transplanted stem cells. Recent reports have identified a class of microvesicles, termed exosomes, released by stem cells that are able to confer therapeutic effects on injured renal and cardiac tissue. In this issue of Stem Cell Research & Therapy, Zhou and colleagues demonstrate the ability of exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs), but not non-stem cell-derived exosomes, to improve acute kidney injury induced by cisplatin in rats. The authors demonstrate that hucMSC exosomes can reduce cisplatin-mediated renal oxidative stress and apoptosis in vivo and increase renal epithelial cell proliferation in culture. These results suggest that stem cell-derived exosomes, which are easy to isolate and safer to use than the parental stem cells, could have significant clinical utility.

Oxidative damage is a well-established driver of aging. Evidence of oxidative stress exists in aged and degenerated discs, but it is unclear how it affects disc metabolism. In this study, we first determined whether oxidative stress negatively impacts disc matrix metabolism using disc organotypic and cell cultures. Mouse disc organotypic culture grown at atmospheric oxygen (20% O(2)) exhibited perturbed disc matrix homeostasis, including reduced proteoglycan synthesis and enhanced expression of matrix metalloproteinases, compared to discs grown at low oxygen levels (5% O(2)). Human disc cells grown at 20% O(2) showed increased levels of mitochondrial-derived superoxide anions and perturbed matrix homeostasis. Treatment of disc cells with the mitochondria-targeted reactive oxygen species (ROS) scavenger XJB-5-131 blunted the adverse effects caused by 20% O(2). Importantly, we demonstrated that treatment of accelerated aging Ercc1(-/Δ) mice, previously established to be a useful in vivo model to study age-related intervertebral disc degeneration (IDD), also resulted in improved disc total glycosaminoglycan content and proteoglycan synthesis. This demonstrates that mitochondrial-derived ROS contributes to age-associated IDD in Ercc1(-/Δ) mice. Collectively, these data provide strong experimental evidence that mitochondrial-derived ROS play a causal role in driving changes linked to aging-related IDD and a potentially important role for radical scavengers in preventing IDD.

Study Design. NF-κB activity was pharmacologically and genetically blocked in an accelerated aging mouse model to mitigate age-related disc degenerative changes.Objective. To study the mediatory role of NF-κB signaling pathway in age-dependent intervertebral disc degeneration.Summary of Background Data. Aging is a major contributor to intervertebral disc degeneration (IDD), but the molecular mechanism behind this process is poorly understood. NF-κB is a family of transcription factors which play a central role in mediating cellular response to damage, stress, and inflammation. Growing evidence implicates chronic NF-κB activation as a culprit in many aging-related diseases, but its role in aging-related IDD has not been adequately explored. We studied the effects of NF-κB inhibition on IDD using a DNA repair-deficient mouse model of accelerated aging (Ercc1 mice) previously been reported to exhibit age-related IDD.Methods. Systemic inhibition of NF-κB activation was achieved either genetically by deletion of one allele of the NF-κB subunit p65 (Ercc1p65 mice) or pharmacologically by chronic intra-peritoneal administration of the Nemo Binding Domain (8K-NBD) peptide to block the formation of the upstream activator of NF-κB, IκB Inducible Kinase (IKK), in Ercc1 mice. Disc cellularity, total proteoglycan content and proteoglycan synthesis of treated mice and untreated controls were assessed.Results. Decreased disc matrix proteoglycan content, a hallmark feature of IDD, and elevated disc NF-κB activity were observed in discs of progeroid Ercc1 mice and naturally aged wild-type compared to young WT mice. Systemic inhibition of NF-κB by the 8K-NBD peptide in Ercc1 mice increased disc proteoglycan synthesis and ameriolated loss disc cellularity and matrix proteoglycan. These results were confirmed genetically by using the p65 haploinsufficient Ercc1p65 mice.Conclusion. These findings demonstrate that the IKK/NF-κB signaling pathway is a key mediator of age-dependent IDD and represents a therapeutic target for mitigating disc degenerative diseases associated with aging.

Local gene transfer of the human LIM Mineralization Protein (LMP), a novel intracellular positive regulator of the osteoblast differentiation program, can induce efficient bone formation in rodents. In order to develop a clinically relevant gene therapy approach to facilitate bone healing, we have used primary dermal fibroblasts transduced ex vivo with Ad.LMP3 and seeded on an hydroxyapatite/collagen matrix prior to autologous implantation. Here we demonstrate that genetically modified autologous dermal fibroblasts expressing Ad.LMP-3 are able to induce ectopic bone formation following implantation of the matrix into the mouse triceps and paravertebral muscles. Moreover, implantation of the Ad.LMP-3-modified dermal fibroblasts into a rat mandibular bone critical size defect model results in efficient healing as determined by X-ray, histology and three dimensional micro computed tomography (3DμCT). These results demonstrate the effectiveness of the non-secreted intracellular osteogenic factor LMP-3, in inducing bone formation in vivo. Moreover, the utilization of autologous dermal fibroblasts implanted on a biomaterial represents a promising approach for possible future clinical applications aimed at inducing new bone formation.

Intervertebral disc degeneration (IDD) is the leading cause of debilitating spinal disorders such as chronic lower back pain. Aging is the greatest risk factor for IDD. Previously, we demonstrated IDD in a murine model of a progeroid syndrome caused by reduced expression of a key DNA repair enzyme. This led us to hypothesize that DNA damage promotes IDD. To test our hypothesis, we chronically exposed adult wild-type (Wt) and DNA repair-deficient Ercc1(-/Δ) mice to the cancer therapeutic agent mechlorethamine (MEC) or ionization radiation (IR) to induce DNA damage and measured the impact on disc structure. Proteoglycan, a major structural matrix constituent of the disc, was reduced 3-5× in the discs of MEC- and IR-exposed animals compared to untreated controls. Expression of the protease ADAMTS4 and aggrecan proteolytic fragments was significantly increased. Additionally, new PG synthesis was reduced 2-3× in MEC- and IR-treated discs compared to untreated controls. Both cellular senescence and apoptosis were increased in discs of treated animals. The effects were more severe in the DNA repair-deficient Ercc1(-/Δ) mice than in Wt littermates. Local irradiation of the vertebra in Wt mice elicited a similar reduction in PG. These data demonstrate that genotoxic stress drives degenerative changes associated with IDD.

Lim mineralization protein-3 (LMP3) induces osteoblast differentiation by regulating the expression and activity of certain molecules involved in the osteogenic cascade, including those belonging to the bone morphogenetic protein (BMP) family. The complete network of molecular events involved in LMP3-mediated osteogenesis is still unknown. The aim of this study was to analyze the genome-wide gene expression profiles in human mesenchymal stem cells (hMSC) induced by exogenous LMP3 to mediate osteogenesis. For this purpose hMSC were transduced with a defective adenoviral vector expressing the human LMP3 gene and microarray analysis was performed 1 day post-adenoviral transduction. Cells transduced with the vector backbone and untransduced cells were used as independent controls in the experiments. Microarray data were independently validated by means of real-time PCR on selected transcripts. The statistical analysis of microarray data produced a list of 263 significantly (p < 0.01) differentially expressed transcripts. The biological interpretation of the results indicated, among the most noteworthy effects, the modulation of genes involved in the TGF-beta1 pathway: 88 genes coding for key regulators of the cell cycle regulatory machinery and 28 genes implicated in the regulation of cell proliferation along with the development of connective, muscular, and skeletal tissues. These results suggested that LMP3 could affect the fine balance between cell proliferation/differentiation of mesenchymal cells mostly by modulating the TGF-beta1 signaling pathway.

Introduction. Gene therapy research in the field of orthopaedics have evolved during the last decade, leading to possible applications for the treatment of pathological conditions, such as bone fractures and defects. Several gene transfer techniques have been employed so far for inducing bone formation in animal models of bone defects. Cell-based approaches, such as the implantation in animals of ex vivo genetically modified cells, produced promising results. In this study we used autologous skin fibroblasts, which are very simple to harvest and propagate in culture, transduced ex vivo with the osteogenic factor Lim Mineralization Protein-3 (LMP-3). These engineered cells produced successful bone healing when implanted by the use of a scaffold in rats, validating the in vivo osteoinductive properties of hLMP-3. Materials and Methods. Primary dermal fibroblasts cultures were established using a biopsy of shaved skin obtained from the abdomen of each rat. Semi-confluent primary fibroblasts were infected with either AdBMP-2 or AdhLMP-3, using a overall multiplicity of infection (MOI) of 100 viral particles per cell. Cells transduced with Ad-eGFP were used as a viral infection control, while untreated cells served as a negative control. The transduced cells were harvested 24 hours after viral infection, let adsorbed on a Hydroxyapatite/Collagen scaffold and then implanted in a bone defect surgically performed in the mandible of immunocompetent rats. The animals were divided in 4 groups: rats treated with cells infected with AdLMP-3, rats treated with cells infected with AdBMP-2, rats treated with cells infected with Ad-eGFP and rats treated with untreated cells. Rats from each group were sacrified at 1, 2 and 3 months after the treatment and studied by x-rays, Micro-CT and histology. Results. All the animals treated with LMP-3 showed healing of the bone defect after 3 months, as confirmed histologically and radiographically. On the contrary none of the controls showed bone formation at the latest time point. Discussion. Recently, Lim Mineralization Proteins (LMP) have been identified as regulators of the osteoblast differentiation program. We have previously demonstrated that human LMP-3 contributes actively to bone formation, acting through the BMP-2 signaling pathway, being capable of inducing differentiation of cells of mesenchymal derivation towards the osteoblastic lineage, through the up-regulation of bone-specific genes, along with ectopic bone formation in vivo and mineralization in vitro. In this study we have tested the efficacy of an ex-vivo approach using autologous dermal fibroblasts infected with AdLMP-3. Our results show that it s possible to induce a complete bone healing using this method, and confirm the in vivo osteoinductive properties of hLMP-3.

Gene therapy has developed during the last two decades as a promising strategy for orthopaedics applications, since several different gene transfer techniques proved to be effective, both in vitro and in vivo, for the induction of bone formation. Successful results have been achieved with gene-based bone healing strategies in several preclinical studies, using different animal models. New genes and new viral and non-viral vector constructs have been developed to reduce the risks and safety issues, widening the field of possible applications and improving the potential therapeutical effects. We review the latest gene transfer technologies employed for in vivo bone formation, focusing on the recently identified network of growth factors and genes involved in the modulation of the osteogenetic process and on the variety of vectors utilized for gene delivery.

LIM mineralization protein (LMP) is a novel positive regulator of the osteoblast differentiation program. In humans, three different LMP splice variants have been identified: LMP-1, LMP-2, and LMP-3. Gene transfer of human LMP-1 (hLMP-1) induces expression of genes involved in bone formation, including certain bone morphogenetic proteins (BMPs), promotes bone nodule formation in vitro, ectopic bone formation in vivo, and is therapeutic in animal models of posterior thoracic and lumbar spine fusion. To examine the osteoinductive properties of the LMP-3 in vitro and in vivo, we have generated plasmid and adenoviral vectors expressing codon-optimized hLMP-3. Here we demonstrate that gene transfer of hLMP-3 induces expression of the bone-specific genes osteocalcin, osteopontin, and bone sialoprotein and induced bone mineralization in preosteoblastic and fibroblastic cells. We also demonstrate that hLMP-3 is able to induce bone mineralization and the expression of the bone-specific genes, BMP-2, OSX, RunX2, and alkaline phosphatase in human mesenchymal stem cells in a dose-dependent manner. Finally, we demonstrate that direct gene transfer of hLMP-3 into murine skeletal muscle results in ectopic bone formation more efficiently than BMP-2. These results demonstrate that hLMP-3 gene transfer can be used to promote bone formation in cell culture and in vivo as or more efficiently than BMP-2, thus establishing feasibility and efficacy of direct gene delivery of hLMP-3 to produce bone in vivo. These results suggest that gene transfer of hLMP-3 could be developed as a bone-inductive therapeutic agent for clinical applications.