1,721,010 research outputs found
In Vivo Results of a Clinically Relevant Gene Therapy Approach for Orthopaedic Applicaions
No full textGene therapy for in vivo bone formation: recent advances
No full textGene 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
Ex Vivo Gene Therapy Approach Using Human Lim Mineralization Protein-3 To Induce Bone Healing in a Rodent Model
No full textIntroduction. 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
Cloning of the Minimal Functional Domain of Human Lim Mineralization Protein-3 Able To Induce Bone Mineralization: In Vitro and In Vivo Study
No full textHuman 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
Efficient bone formation by gene transfer of human LIM mineralization protein-3
No full textLIM 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
Ex Vivo Gene Therapy Approach Using Human Lim Mineralization Protein-3 To Induce Bone Healing in a Rodent.
No full textRisultati pre-clinici di un nuovo approccio di terapia genica ex-vivo: fusioni vertebrale e guarigione delle “non-union fractures” tramite l’impianto di fibroblasti autologhi geneticamente modificati per l’espressione di HLMP-3. Volume 8, Supplement 1, Novembre
No full textEx Vivo Gene Therapy Approach Using Human Lim Mineralization Protein-3 To Induce Bone Healing in a Rodent Model
No full textIntroduction. 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
Early Transcriptional Events During Osteogenic Differentiation of Human Bone Marrow Stromal Cells Induced by Lim Mineralization Protein 3
No full textLim 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
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