Toxic effects of local anesthetics on rat fibroblasts: An in-vitro study.

Background: Infusion catheters facilitate a controlled infusion of local anesthetic (LA) for pain control after surgery. However, their potential effects on healing fibroblasts are unspecified.

Methods: Rat synovial fibroblasts were cultured in 12-well plates.

Gene Therapy in Orthopaedics: Progress and Challenges in Pre-Clinical Development and Translation.

In orthopaedics, gene-based treatment approaches are being investigated for an array of common -yet medically challenging- pathologic conditions of the skeletal connective tissues and structures (bone, cartilage, ligament, tendon, joints, intervertebral discs etc.). As the skeletal system protects the vital organs and provides weight-bearing structural support, the various tissues are principally composed of dense extracellular matrix (ECM), often with minimal cellularity and vasculature.

Gene Therapy for Osteoarthritis: Pharmacokinetics of Intra-Articular Self-Complementary Adeno-Associated Virus Interleukin-1 Receptor Antagonist Delivery in an Equine Model.

Toward the treatment of osteoarthritis (OA), the authors have been investigating self-complementary adeno-associated virus (scAAV) for intra-articular delivery of therapeutic gene products. As OA frequently affects weight-bearing joints, pharmacokinetic studies of scAAV gene delivery were performed in the joints of the equine forelimb to identify parameters relevant to clinical translation in humans. Using interleukin-1 receptor antagonist (IL-1Ra) as a secreted therapeutic reporter, scAAV vector plasmids containing codon-optimized cDNA for equine IL-1Ra (eqIL-1Ra) were generated, which produced eqIL-1Ra at levels 30- to 50-fold higher than the native sequence.

Self-Complementary Adeno-Associated Virus-Mediated Interleukin-1 Receptor Antagonist Gene Delivery for the Treatment of Osteoarthritis: Test of Efficacy in an Equine Model.

The authors are investigating self-complementary adeno-associated virus (scAAV) as a vector for intra-articular gene-delivery of interleukin-1 receptor antagonist (IL-1Ra), and its therapeutic capacity in the treatment of osteoarthritis (OA). To model gene transfer on a scale proportional to the human knee, a frequent site of OA incidence, studies were focused on the joints of the equine forelimb. Using AAV2.

Evidence for the osteosarcoma stem cell.

Osteosarcoma is a highly malignant bone tumor of children and young adults. Cytotoxic chemotherapy combined with aggressive surgery only has a 60% survival rate. Historically, chemotherapy has been developed assuming that all cells within a particular cancer are clonal and near identical.

Gene delivery of TGF-β1 induces arthrofibrosis and chondrometaplasia of synovium in vivo.

To understand the cellular and molecular events contributing to arthrofibrosis, we used an adenovirus to deliver and overexpress transforming growth factor-beta 1 (TGF-β1) cDNA (Ad.TGF-β1) in the knee joints of immunocompromised rats. Following delivery, animals were killed periodically, and joint tissues were examined macroscopically and histologically.

Expression of an exogenous human Oct-4 promoter identifies tumor-initiating cells in osteosarcoma.

We explored the nature of the tumor-initiating cell in osteosarcoma, a bone malignancy that predominately occurs in children. Previously, we observed expression of Oct-4, an embryonal transcriptional regulator, in osteosarcoma cell cultures and tissues. To examine the relationship between Oct-4 and tumorigenesis, cells from an osteosarcoma biopsy (OS521) were stably transfected with a plasmid containing the human Oct-4 promoter driving a green fluorescent protein (GFP) reporter to generate the transgenic line OS521Oct-4p.

Intra-articular gene delivery and expression of interleukin-1Ra mediated by self-complementary adeno-associated virus.

Background: The adeno-associated virus (AAV) has many safety features that favor its use in the treatment of arthritic conditions; however, the conventional, single-stranded vector is inefficient for gene delivery to fibroblastic cells that primarily populate articular tissues. This has been attributed to the inability of these cells to convert the vector to a double-stranded form. To overcome this, we evaluated double-stranded self-complementary (sc) AAV as a vehicle for intra-articular gene delivery.

Perspectives on the use of gene therapy for chronic joint diseases.

Advances in molecular and cellular biology have identified a wide variety of proteins including targeted cytokine inhibitors, immunomodulatory proteins, cytotoxic mediators, angiogenesis inhibitors, and intracellular signalling molecules that could be of great benefit in the treatment of chronic joint diseases, such as osteo- and rheumatoid arthritis. Unfortunately, protein-based drugs are difficult to administer effectively. They have a high rate of turnover, requiring frequent readministration, and exposure in non-diseased tissue can lead to serious side effects.

Exogenous glucosamine globally protects chondrocytes from the arthritogenic effects of IL-1beta.

The effects of exogenous glucosamine on the biology of articular chondrocytes were determined by examining global transcription patterns under normal culture conditions and following challenge with IL-1beta. Chondrocytes isolated from the cartilage of rats were cultured in several flasks either alone or in the presence of 20 mM glucosamine. Six hours later, one-half of the cultures of each group were challenged with 10 ng/ml IL-1beta.

Recruitment of transcription complexes to the beta-globin locus control region and transcription of hypersensitive site 3 prior to erythroid differentiation of murine embryonic stem cells.

Eukaryotic chromosomal DNA is densely packaged in the nucleus and organized into discrete domains of active and inactive chromatin. Gene loci that are activated during the process of cell differentiation undergo changes that result in modifications of specific histone tail residues and in loosening of chromatin structure. The beta-globin genes are expressed exclusively in erythroid cells.

Locus control region elements HS2 and HS3 in combination with chromatin boundaries confer high-level expression of a human beta-globin transgene in a centromeric region.

Expression constructs are subject to position-effects in transgenic assays unless they harbour elements that protect them from negative or positive influences exerted by chromatin at the site of integration. Locus control regions (LCRs) and boundary elements are able to protect from position effects by preventing heterochromatization of linked genes. The LCR in the human beta-globin gene locus is located far upstream of the genes and composed of several erythroid specific DNase I hypersensitive (HS) sites.

Recruitment of transcription complexes to the beta-globin gene locus in vivo and in vitro.

Erythroid-specific, high level expression of the beta-globin genes is regulated by the locus control region (LCR), composed of multiple DNase I-hypersensitive sites and located far upstream of the genes. Recent studies have shown that LCR core elements recruit RNA polymerase II (pol II). In the present study we demonstrate the following: 1) pol II and other basal transcription factors are recruited to LCR core hypersensitive elements; 2) pol II dissociates from and re-associates with the globin gene locus during replication; 3) pol II interacts with the LCR but not with the beta-globin gene prior to erythroid differentiation in embryonic stem cells; and 4) the erythroid transcription factor NF-E2 facilitates the transfer of pol II from immobilized LCR constructs to a beta-globin gene in vitro.

The human beta-globin locus control region.

The human beta-globin gene locus is the subject of intense study, and over the past two decades a wealth of information has accumulated on how tissue-specific and stage-specific expression of its genes is achieved. The data are extensive and it would be difficult, if not impossible, to formulate a comprehensive model integrating every aspect of what is currently known. In this review, we introduce the fundamental characteristics of globin locus regulation as well as questions on which much of the current research is predicated.