scholarly journals Lentivirus-mediated gene transfer induces long-term transgene expression of BMP-2 in vitro and new bone formation in vivo

2005 ◽  
Vol 11 (3) ◽  
pp. 390-398 ◽  
Author(s):  
Osamu Sugiyama ◽  
Dong Sung An ◽  
Sam P.K. Kung ◽  
Brian T. Feeley ◽  
Seth Gamradt ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Bone Formation ◽  
Transgene Expression ◽  
New Bone Formation

In Vivo and In Vitro Evaluation of Coated HAp Films with Different Surface Morphology

2007 ◽  
Vol 539-543 ◽  
pp. 710-715
Author(s):  
Kotaro Kuroda ◽  
Ryoichi Ichino ◽  
Masazumi Okido
Keyword(s):  
Bone Formation ◽  
Surface Morphology ◽  
New Bone Formation ◽  
Contact Ratio ◽  
Bone Implant ◽  
Ft Ir ◽  
Mouse Osteoblast ◽  
Surface Morphologies

Hydroxyapatite (HAp) coatings were formed on cp titanium plates and rods by the thermal substrate method in an aqueous solution that included 0.3 mM Ca(H2PO4)2 and 0.7 mM CaCl2. The coating experiments were conducted at 40-140 oC and pH = 8 for 15 or 30 min. The properties for the coated samples were studied using XRD, EDX, FT-IR, and SEM. All the specimens were covered with HAp, which had different surface morphologies such as net-like, plate-like and needle-like. After cleaning and sterilization, all the coated specimens were subjected to in vivo and vitro testing. In the in vitro testing, the mouse osteoblast-like cells (MC3T3-E1) were cultured on the coated and non-coated specimens for up to 30 days. Moreover, the specimens (φ2 x 5 mm) were implanted in rats femoral for up to 8 weeks, the osseoinductivity on them were evaluated. In in vitro evaluations, there were not significant differences between the different surface morphologies. In in vivo evaluations, however, two weeks postimplantation, new bone formed on both the HAp coated and non-coated titanium rods in the cancellous and cortical bone. The bone-implant contact ratio, which was used for the evaluation of new bone formation, was significantly dependent on the surface morphology of the HAp, and the results demonstrated that the needle-like coating appears to promote rapid bone formation.

In Vitro and In Vivo Evaluation Of Calcium Phosphate Bone Graft Substitutes

2010 ◽  
Vol 654-656 ◽  
pp. 2065-2070
Author(s):  
Ho Yeon Song ◽  
Young Hee Kim ◽  
Jyoti M. Anirban ◽  
In Seon Byun ◽  
Kyung A Kwak ◽  
...  
Keyword(s):  
Calcium Phosphate ◽  
Bone Formation ◽  
Bone Graft ◽  
Cellular Proliferation ◽  
New Bone Formation ◽  
Hydroxy Apatite ◽  
Defect Sites ◽  
Bone Graft Substitutes

Calcium phosphate ceramics such as hydroxy apatite (HA), β-tricalcium phosphate (β-TCP) and bicalcium phosphate (BCP) have been used as a bone graft biomaterial because of their good biocompatibility and similarity of chemical composition to natural bones. To increase the mechanical and osteoconductive properties, the granules and spongy type porous bone graft substitutes were prepared by fibrous monolithic process and polyurethane foam replica methods, respectively. The pore sizes obtained using these approaches ranged between 100-600 µm. The cytotoxicity, cellular proliferation, differentiation and ECM deposition on the bone graft substitutes were observed by SEM and confocal microscopy. Moreover, the scaffolds were implanted in the rabbit femur. New bone formation and biodegradation of bone graft were observed through follow-up X-ray, micro-CT analysis and histological findings. After several months (2, 3, 6, 12 and 24 months) of implantation, new bone formation and ingrowths were observed in defect sites of the animal by CaP ceramics and 2 to 3 times higher bone ingrowths were confirmed than that of the normal trabecular bones in terms of total bone volume (BV).

Clonal Analyses of Retroviral Vector Integrations in ADA-SCID Patients Treated with Stem Cell Gene Therapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3249-3249
Author(s):  
Barbara Cassani ◽  
Grazia Andolfi ◽  
Massimiliano Mirolo ◽  
Luca Biasco ◽  
Alessandra Recchia ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Human Genome ◽  
Ex Vivo ◽  
Cd34 Cells

Abstract Gene transfer into hematopoietic stem/progenitor cells (HSC) by gammaretroviral vectors is an effective treatment for patients affected by severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA)-deficiency. Recent studied have indicated that gammaretroviral vectors integrate in a non-random fashion in their host genome, but there is still limited information on the distribution of retroviral insertion sites (RIS) in human long-term reconstituting HSC following therapeutic gene transfer. We performed a genome-wide analysis of RIS in transduced bone marrow-derived CD34+ cells before transplantation (in vitro) and in hematopoietic cell subsets (ex vivo) from five ADA-SCID patients treated with gene therapy combined to low-dose busulfan. Vector-genome junctions were cloned by inverse or linker-mediated PCR, sequenced, mapped onto the human genome, and compared to a library of randomly cloned human genome fragments or to the expected distribution for the NCBI annotation. Both in vitro (n=212) and ex vivo (n=496) RIS showed a non-random distribution, with strong preference for a 5-kb window around transcription start sites (23.6% and 28.8%, respectively) and for gene-dense regions. Integrations occurring inside the transcribed portion of a RefSeq genes were more represented in vitro than ex vivo (50.9 vs 41.3%), while RIS <30kb upstream from the start site were more frequent in the ex vivo sample (25.6% vs 19.4%). Among recurrently hit loci (n=50), LMO2 was the most represented, with one integration cloned from pre-infusion CD34+ cells and five from post-gene therapy samples (2 in granulocytes, 3 in T cells). Clone-specific Q-PCR showed no in vivo expansion of LMO2-carrying clones while LMO2 gene overexpression at the bulk level was excluded by RT-PCR. Gene expression profiling revealed a preference for integration into genes transcriptionally active in CD34+ cells at the time of transduction as well as genes expressed in T cells. Functional clustering analysis of genes hit by retroviral vectors in pre- and post-transplant cells showed no in vivo skewing towards genes controlling self-renewal or survival of HSC (i.e. cell cycle, transcription, signal transduction). Clonal analysis of long-term repopulating cells (>=6 months) revealed a high number of distinct RIS (range 42–121) in the T-cell compartment, in agreement with the complexity of the T-cell repertoire, while fewer RIS were retrieved from granulocytes. The presence of shared integrants among multiple lineages confirmed that the gene transfer protocol was adequate to allow stable engraftment of multipotent HSC. Taken together, our data show that transplantation of ADA-transduced HSC does not result in skewing or expansion of malignant clones in vivo, despite the occurrence of insertions near potentially oncogenic genomic sites. These results, combined to the relatively long-term follow-up of patients, indicate that retroviral-mediated gene transfer for ADA-SCID has a favorable safety profile.

Biological Reaction to Electrically Polarized Hydroxyapatite

2000 ◽  
Vol 662 ◽  
Author(s):  
Tomoko Sakai ◽  
Masato Ueshima ◽  
Sadao Morita ◽  
Satoshi Nakamura ◽  
Kimihiro Yamashita
Keyword(s):  
Bone Formation ◽  
Body Fluid ◽  
Bone Defects ◽  
New Bone Formation ◽  
In Vitro Experiment ◽  
In Vivo Experiments ◽  
Biological Reaction ◽  
Positively Charged

AbstractWe have studied the polarized hydroxyapatite (HAp) whose surface was negatively or positively charged. In this study, we assessed the interfaces in vitro and in vivo periodically. As in vitro experiment, samples were immersed in simulated body fluid for 7 days and the surface was examined by scanning electron microscope (SEM). As in vivo experiments, cortical bone defects were created on the femoral trochanters and the condyles of the six Japanese white rabbits and the samples were implanted. The rabbits were sacrificed at 1, 2 and 4 W after the operation to analyze the surfaces by the SEM and the optical microscopy. In this study, a new thick apatite layer was formed on the negatively charged surface (N-surface) after 1week immersion in SBF in vitro. Besides, significant new bone formation was found at 2 weeks after the operation on N-surface in vivo, which was earlier than positively charged or non-polarized HAp surface. From this study negatively charged HAp surface by polarization accelerated the HAp crystal growth or the new bone formation. Thus, this N-surface will be promising for earlier fixation of the prosthesis or better recovery of the bone defect.

The potential of stromal cell-derived factor-1 delivery using a collagen membrane for bone regeneration

2017 ◽  
Vol 31 (7) ◽  
pp. 1049-1061 ◽  
Author(s):  
Tadahiro Takayama ◽  
Jisen Dai ◽  
Keita Tachi ◽  
Ryutaro Shohara ◽  
Hironori Kasai ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Growth Factor ◽  
Bone Formation ◽  
Bone Regeneration ◽  
Stromal Cell ◽  
In Vitro Studies ◽  
New Bone Formation ◽  
Stromal Cell Derived Factor

Stromal cell-derived factor-1 (SDF-1) is a cytokine that is important in stem and progenitor cell recruitment in tissue repair after injury. Regenerative procedures using collagen membranes (CMs) are presently well established in periodontal and implant dentistry. The objective of this study is to test the subsequent effects of the released SDF-1 from a CM on bone regeneration compared to platelet-derived growth factor (PDGF) in vitro and in vivo. For in vitro studies, cell proliferation, alkaline phosphatase activity, and osteoblastic differentiation marker genes were assessed after MC3T3-E1 mouse preosteoblasts were cultured with CMs containing factors. In vivo effects were investigated by placement of CMs containing SDF-1 or PDGF using a rat mandibular bone defect model. At 4 weeks after the surgery, the new bone formation was measured using micro-computed tomography (µCT) and histological analysis. The results of in vitro studies revealed that CM delivery of SDF-1 significantly induced cell proliferation, ALP activity, and gene expression of all osteogenic markers compared to the CM alone or control, similar to PDGF. Quantitative and qualitative µCT analysis for volume of new bone formation and the percentage of new bone area showed that SDF-1-treated groups significantly increased and accelerated bone regeneration compared to control and CM alone. The enhancement of bone formation in SDF-1-treated animals was dose-dependent and with levels similar to those measured with PDGF. These results suggest that a CM with SDF-1 may be a great candidate for growth factor delivery that could be a substitute for PDGF in clinical procedures where bone regeneration is necessary.

In vivo administration of lentiviral vectors triggers a type I interferon response that restricts hepatocyte gene transfer and promotes vector clearance

Blood ◽  
2006 ◽  
Vol 109 (7) ◽  
pp. 2797-2805 ◽  
Author(s):  
Brian D. Brown ◽  
Giovanni Sitia ◽  
Andrea Annoni ◽  
Ehud Hauben ◽  
Lucia Sergi Sergi ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Transgene Expression ◽  
Lentiviral Vectors ◽  
Interferon Response ◽  
Type I ◽  
Nonparenchymal Cells ◽  
Antiviral Responses ◽  
Liver Gene

AbstractLiver gene transfer is a highly sought goal for the treatment of inherited and infectious diseases. Lentiviral vectors (LVs) have many desirable properties for hepatocyte-directed gene delivery, including the ability to integrate into nondividing cells. Unfortunately, upon systemic administration, LV transduces hepatocytes relatively inefficiently compared with nonparenchymal cells, and the duration of transgene expression is often limited by immune responses. Here, we investigated the role of innate antiviral responses in these events. We show that administration of LVs to mice triggers a rapid and transient IFNαβ response. This effect was dependent on functional vector particles, and in vitro challenge of antigen-presenting cells suggested that plasmacytoid dendritic cells initiated the response. Remarkably, when LVs were administered to animals that lack the capacity to respond to IFNαβ, there was a dramatic increase in hepatocyte transduction, and stable transgene expression was achieved. These findings indicate that, even in the setting of acute delivery of replication-defective vectors, IFNs effectively interfere with transduction in a cell-type–specific manner. Moreover, because disabling a single component of the innate/immune network was sufficient to establish persistent xenoantigen expression, our results raise the hope that the immunologic barriers to gene therapy are less insurmountable than expected.

scholarly journals Long-Term Transgene Expression in Proliferating Cells Mediated by Episomally Maintained High-Capacity Adenovirus Vectors

2004 ◽  
Vol 78 (1) ◽  
pp. 9-22 ◽  
Author(s):  
Florian Kreppel ◽  
Stefan Kochanek
Keyword(s):  
Transgene Expression ◽  
High Capacity ◽  
Target Cells ◽  
Resting Cells ◽  
Proliferating Cells ◽  
Daughter Cells ◽  
Adenovirus Vectors

ABSTRACT High-capacity “gutless” adenovirus vectors (HC-AdV) mediate long-term transgene expression in resting cells in vitro and in vivo because of low toxicity and immunogenicity. However, in proliferating cells, expression is transient since HC-AdV genomes do not possess elements that allow for replication and segregation of the replicated genomes to daughter cells. We developed a binary HC-AdV system that, under certain conditions, allows for significantly prolonged episomal maintenance of HC-AdV genomes in proliferating tissue culture cells, resulting in sustained transgene expression. After transduction of target cells the linear HC-AdV genomes were circularized by the DNA recombinase FLPe, which was expressed from the second HC-AdV. The oriP/EBNA-1 replication system derived from Epstein-Barr virus, as well as the human replication origin from the lamin B2 locus, were used as cis elements to test for replication of the 28-kb circular vector genomes with or without selective pressure. Depending on the system, up to 98% of the circularized genomes were replicated and segregated to daughter cells, as demonstrated by Southern assays and as confirmed by monitoring EGFP transgene expression. Surprisingly, in the absence of FLPe recombinase, a small but significant number of HC-AdV genomes spontaneously circularized after transduction of target cells. These circles, found to contain end-to-end joined adenovirus termini, replicated with increased efficiency compared to vectors circularized by FLPe. After further improvements, this HC-AdV system might be suitable for gene therapy applications requiring long-term transgene expression.

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