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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. Dilutions were prepared in a solution containing reduced-serum media and 0.9% sodium chloride in 1:1 concentration. Each well was treated with 500 µl of the appropriate LA dilution or normal saline for 15- or 30-min. LA dilutions included: 0.5% ropivacaine HCl, 0.2% ropivacaine HCl, 1% lidocaine HCl and epinephrine 1:100,000, 1% lidocaine HCl, 0.5% bupivacaine HCl and epinephrine 1:200,000, and 0.5% bupivacaine HCl. This was replicated three times. Dilution of each LA whereby 50% of the cells were unviable (Lethal dose 50 [LD50]) was analyzed. RESULTS: LD50 was reached for lidocaine and bupivacaine, but not ropivacaine. Lidocaine 1% with epinephrine is toxic at 30-min at 1/4 and 1/2 sample dilutions. Bupivacaine 0.5% was found to be toxic at 30-min at 1/2 sample dilution. Bupivacaine 0.5% with epinephrine was found to be toxic at 15- and 30-min at 1/4 sample dilution. Lidocaine 1% was found to be toxic at 15- and 30-min at 1/2 sample dilution. Ropivacaine 0.2% and 0.5% remained below LD50 at all time-points and concentrations, with 0.2% demonstrating the least cell death. CONCLUSIONS: Though pain pumps are generally efficacious, LAs may inhibit fibroblasts, including perineural fibroblast and endoneurial fibroblast-like cells, which may contribute to persistent nerve deficits, delayed neurogenic pain, and negatively impact healing. Should a continuous infusion be used, our data supports ropivacaine 0.2%. LEVEL OF EVIDENCE: Basic Science Study; Animal model.
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PURPOSE OF REVIEW: To assess the present status of gene therapy for osteoarthritis (OA). RECENT FINDINGS: An expanding list of cDNAs show therapeutic activity when introduced into the joints of animals with experimental models of OA. In vivo delivery with adenovirus or adeno-associated virus is most commonly used for this purpose. The list of encoded products includes cytokines, cytokine antagonists, enzymes, enzyme inhibitors, growth factors and noncoding RNA. Elements of CRISPR-Cas have also been delivered to mouse knees to ablate key genes. Several human trials have been initiated, using transgenes encoding transforming growth factor-ß1, interleukin-1 receptor antagonist, interferon-ß, the NKX3.2 transcription factor or variant interleukin-10. The first of these, using ex vivo delivery with allogeneic chondrocytes, gained approval in Korea which was subsequently retracted. However, it is undergoing Phase III clinical trials in the United States. The other trials are in Phase I or II. No gene therapy for OA has current marketing approval in any jurisdiction. SUMMARY: Extensive preclinical data support the use of intra-articular gene therapy for treating OA. Translation is beginning to accelerate and six gene therapeutics are in clinical trials. Importantly, venture capital has begun to flow and at least seven companies are developing products. Significant progress in the future can be expected.
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Cartílago Articular , Osteoartritis , Humanos , Ratones , Animales , Osteoartritis/terapia , Osteoartritis/tratamiento farmacológico , Terapia Genética , Condrocitos/metabolismo , Proteína Antagonista del Receptor de Interleucina 1/uso terapéutico , Citocinas/metabolismo , Cartílago Articular/metabolismoRESUMEN
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. Due to their functional roles, composition, and distribution throughout the body the skeletal tissues are prone to traumatic injury, and/or structural failure from chronic inflammation and matrix degradation. Due to a mixture of environment and endogenous factors repair processes are often slow and fail to restore the native quality of the ECM and its function. In other cases, large-scale lesions from severe trauma or tumor surgery, exceed the body's healing and regenerative capacity. Although a wide range of exogenous gene products (proteins and RNAs) have the potential to enhance tissue repair/regeneration and inhibit degenerative disease their clinical use is hindered by the absence of practical methods for safe, effective delivery. Cumulatively, a large body of evidence demonstrates the capacity to transfer coding sequences for biologic agents to cells in the skeletal tissues to achieve prolonged delivery at functional levels to augment local repair or inhibit pathologic processes. With an eye toward clinical translation, we discuss the research progress in the primary injury and disease targets in orthopaedic gene therapy. Technical considerations important to the exploration and pre-clinical development are presented, with an emphasis on vector technologies and delivery strategies whose capacity to generate and sustain functional transgene expression in vivo is well-established.
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Detection of the SARS-CoV-2 spike protein and inactivated virus was achieved using disposable and biofunctionalized functional strips, which can be connected externally to a reusable printed circuit board for signal amplification with an embedded metal-oxide-semiconductor field-effect transistor (MOSFET). A series of chemical reactions was performed to immobilize both a monoclonal antibody and a polyclonal antibody onto the Au-plated electrode used as the sensing surface. An important step in the biofunctionalization, namely, the formation of Au-plated clusters on the sensor strips, was verified by scanning electron microscopy, as well as electrical measurements, to confirm successful binding of thiol groups on this Au surface. The functionalized sensor was externally connected to the gate electrode of the MOSFET, and synchronous pulses were applied to both the sensing strip and the drain contact of the MOSFET. The resulting changes in the dynamics of drain waveforms were converted into analog voltages and digital readouts, which correlate with the concentration of proteins and virus present in the tested solution. A broad range of protein concentrations from 1 fg/ml to 10 µg/ml and virus concentrations from 100 to 2500 PFU/ml were detectable for the sensor functionalized with both antibodies. The results show the potential of this approach for the development of a portable, low-cost, and disposable cartridge sensor system for point-of-care detection of viral diseases.
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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 AAV2.5 capsid and equine IL-1Ra as a homologous transgene, a functional ceiling dose of â¼5 × 1012 viral genomes was previously identified, which elevated the steady state levels of eqIL-1Ra in synovial fluids by >40-fold over endogenous production for at least 6 months. Here, using an osteochondral fragmentation model of early OA, the functional capacity of scAAV.IL-1Ra gene-delivery was examined in equine joints over a period of 12 weeks. In the disease model, transgenic eqIL-1Ra expression was several fold higher than seen previously in healthy joints, and correlated directly with the severity of joint pathology at the time of treatment. Despite wide variation in expression, the steady-state eqIL-1Ra in synovial fluids exceeded that of IL-1 by >400-fold in all animals, and a consistent treatment effect was observed. This included a 30-40% reduction in lameness and â¼25% improvement in total joint pathology by both magnetic resonance imaging and arthroscopic assessments, which included reduced joint effusion and synovitis, and improved repair of the osteochondral lesion. No vector-related increase in eqIL-1Ra levels in blood or urine was noted. Cumulatively, these studies in the equine model indicate scAAV.IL-1Ra administration is reasonably safe and capable of sustained therapeutic IL-1Ra production intra-articularly in joints of human scale. This profile supports consideration for human testing in OA.
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Terapia Genética , Vectores Genéticos/administración & dosificación , Proteína Antagonista del Receptor de Interleucina 1/genética , Osteoartritis/terapia , Animales , Dependovirus/genética , Modelos Animales de Enfermedad , Técnicas de Transferencia de Gen/efectos adversos , Vectores Genéticos/efectos adversos , Vectores Genéticos/genética , Caballos , Humanos , Inyecciones Intraarticulares , Proteína Antagonista del Receptor de Interleucina 1/administración & dosificación , Rodilla/patología , Osteoartritis/genética , Osteoartritis/patologíaRESUMEN
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. The most efficient cDNA was packaged in AAV2.5 capsid, and following characterization in vitro, the virus was injected into the carpal and metacarpophalangeal joints of horses over a 100-fold dose range. A putative ceiling dose of 5 × 1012 viral genomes was identified that elevated the steady-state eqIL-1Ra in the synovial fluids of injected joints by >40-fold over endogenous levels and was sustained for at least 6 months. No adverse effects were seen, and eqIL-1Ra in serum and urine remained at background levels throughout. Using the 5 × 1012 viral genome dose of scAAV, and green fluorescent protein as a cytologic marker, the local and systemic distribution of vector and transduced cells following intra-articular injection scAAV.GFP were compared in healthy equine joints and in those with late-stage, naturally occurring OA. In both cases, 99.7% of the vector remained within the injected joint. Strikingly, the pathologies characteristic of OA (synovitis, osteophyte formation, and cartilage erosion) were associated with a substantial increase in transgenic expression relative to tissues in healthy joints. This was most notable in regions of articular cartilage with visible damage, where foci of brilliantly fluorescent chondrocytes were observed. Overall, these data suggest that AAV-mediated gene transfer can provide relatively safe, sustained protein drug delivery to joints of human proportions.
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Técnicas de Transferencia de Gen , Terapia Genética , Proteína Antagonista del Receptor de Interleucina 1/genética , Osteoartritis/terapia , Animales , Dependovirus/genética , Modelos Animales de Enfermedad , Vectores Genéticos/administración & dosificación , Vectores Genéticos/efectos adversos , Vectores Genéticos/genética , Caballos , Humanos , Inyecciones Intraarticulares , Proteína Antagonista del Receptor de Interleucina 1/administración & dosificación , Osteoartritis/genética , Osteoartritis/patologíaRESUMEN
Musculoskeletal conditions are a major public health problem. Approximately 66 million individuals seek medical attention for a musculoskeletal injury in the United States, with current medical costs being estimated at $873 billion annually. Despite advances in pharmaceuticals, implant materials and surgical techniques, there remains an unmet clinical need for successful treatment of challenging musculoskeletal injuries and pathologic conditions, particularly in the setting of compromised biological environments. Tissue engineering via gene therapy attempts to provide an alternative treatment strategy to address the deficits associated with conventional approaches. The transfer of specific target genes coding for proteins with therapeutic or regenerative properties to target cells and tissues in the disease environment allows for their sustained production and release specifically at the site of interest. The increasing reports of success with gene therapy-based treatments in the clinical management of a variety of diseases provide genuine optimism that similar methods can be adapted for mainstream clinical application in musculoskeletal disorders. In preclinical studies, gene therapy has been successfully used to treat cartilaginous, bone, skeletal muscle, tendon, ligament and intervertebral disk injuries. In addition, gene therapy is being assessed in clinical trials for its safety and therapeutic potential in osteoarthritis. This review will specifically address the clinical potential, preclinical data and future hurdles for gene therapy to be a viable clinical entity for the treatment of fracture nonunion and difficult bone repair scenarios, articular cartilage repair and osteoarthritis.
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Desarrollo Óseo , Enfermedades Óseas/terapia , Cartílago Articular/citología , Terapia Genética , Ingeniería de Tejidos/métodos , Animales , Enfermedades de los Cartílagos/terapia , Curación de Fractura , Humanos , OrtopediaRESUMEN
Most forms of arthritis are incurable, difficult to treat, and a major cause of disability in Western countries. Better local treatment of arthritis is impaired by the pharmacokinetics of the joint that make it very difficult to deliver drugs to joints at sustained, therapeutic concentrations. This is especially true of biologic drugs, such as proteins and RNA, many of which show great promise in preclinical studies. Gene transfer provides a strategy for overcoming this limitation. The basic concept is to deliver cDNAs encoding therapeutic products by direct intra-articular injection, leading to sustained, endogenous synthesis of the gene products within the joint. Proof of concept has been achieved for both in vivo and ex vivo gene delivery using a variety of vectors, genes, and cells in several different animal models. There have been a small number of clinical trials for rheumatoid arthritis (RA) and osteoarthritis (OA) using retrovirus vectors for ex vivo gene delivery and adeno-associated virus (AAV) for in vivo delivery. AAV is of particular interest because, unlike other viral vectors, it is able to penetrate deep within articular cartilage and transduce chondrocytes in situ. This property is of particular importance in OA, where changes in chondrocyte metabolism are thought to be fundamental to the pathophysiology of the disease. Authorities in Korea have recently approved the world's first arthritis gene therapy. This targets OA by the injection of allogeneic chondrocytes that have been transduced with a retrovirus carrying transforming growth factor-ß1 cDNA. Phase III studies are scheduled to start in the United States soon. Meanwhile, two additional Phase I trials are listed on Clinicaltrials.gov , both using AAV. One targets RA by transferring interferon-ß, and the other targets OA by transferring interleukin-1 receptor antagonist. The field is thus gaining momentum and promises to improve the treatment of these common and debilitating diseases.
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Artritis Reumatoide , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Osteoartritis , Animales , Artritis Reumatoide/genética , Artritis Reumatoide/terapia , Dependovirus/genética , Vectores Genéticos , Humanos , Inyecciones Intraarticulares , Modelos Animales , Osteoartritis/genética , Osteoartritis/terapia , Retroviridae/genética , TransgenesRESUMEN
Osteosarcoma (OS) is the most common non-hematologic primary tumor of bone in children and adults. High-dose cytotoxic chemotherapy and surgical resection have improved prognosis, with long-term survival for non-metastatic disease approaching 70%. However, most OS tumors are high grade and tend to rapidly develop pulmonary metastases. Despite clinical advances, patients with metastatic disease or relapse have a poor prognosis. Toward a better understanding of the molecular pathogenesis of human OS, several genetically modified OS mouse models have been developed and will be reviewed here. However, better animal models that more accurately recapitulate the natural progression of the disease are needed for the development of improved prognostic and diagnostic markers as well as targeted therapies for both primary and metastatic OS.
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Arthritis is a disease of joints. The biology of joints makes them very difficult targets for drug delivery in a manner that is specific and selective. This is especially true for proteinaceous drugs ("biologics"). Gene transfer is the only technology that can solve the delivery problem in a clinically reasonable fashion. There is an abundance of preclinical data confirming that genes can be efficiently transferred to tissues within joints by intra-articular injection using a variety of different vectors in conjunction with ex vivo and in vivo strategies. Using the appropriate gene transfer technologies, long-term, intra-articular expression of anti-arthritic transgenes at therapeutic concentrations can be achieved. Numerous studies confirm that gene therapy is effective in treating experimental models of rheumatoid arthritis (RA) and osteoarthritis (OA) in the laboratory. A limited number of clinical trials have been completed, which confirm safety and feasibility but only 3 protocols have reached phase II; as yet, there is no unambiguous evidence of efficacy in human disease. Only 2 clinical trials are presently underway, both phase II studies using allogeneic chondrocytes expressing transforming growth factor-ß1 for the treatment of OA. Phase I studies using adeno-associated virus to deliver interleukin-1Ra in OA and interferon-ß in RA are going through the regulatory process. It is to be hoped that the recent successes in treating rare, Mendelian diseases by gene therapy will lead to accelerated development of genetic treatments for common, non-Mendelian diseases, such as arthritis.
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Artritis/genética , Artritis/terapia , Terapia Genética , Investigación Biomédica Traslacional , Animales , Ensayos Clínicos como Asunto , Técnicas de Transferencia de Gen , Humanos , Articulaciones/patologíaRESUMEN
INTRODUCTION: To date, no single most-appropriate factor or delivery method has been identified for the purpose of mesenchymal stem cell (MSC)-based treatment of cartilage injury. Therefore, in this study we tested whether gene delivery of the growth factor Indian hedgehog (IHH) was able to induce chondrogenesis in human primary MSCs, and whether it was possible by such an approach to modulate the appearance of chondrogenic hypertrophy in pellet cultures in vitro. METHODS: First-generation adenoviral vectors encoding the cDNA of the human IHH gene were created by cre-lox recombination and used alone or in combination with adenoviral vectors, bone morphogenetic protein-2 (Ad.BMP-2), or transforming growth factor beta-1 (Ad.TGF-ß1) to transduce human bone-marrow derived MSCs at 5 × 10² infectious particles/cell. Thereafter, 3 × 105 cells were seeded into aggregates and cultured for 3 weeks in serum-free medium, with untransduced or marker gene transduced cultures as controls. Transgene expressions were determined by ELISA, and aggregates were analysed histologically, immunohistochemically, biochemically and by RT-PCR for chondrogenesis and hypertrophy. RESULTS: IHH, TGF-ß1 and BMP-2 genes were equipotent inducers of chondrogenesis in primary MSCs, as evidenced by strong staining for proteoglycans, collagen type II, increased levels of glycosaminoglycan synthesis, and expression of mRNAs associated with chondrogenesis. IHH-modified aggregates, alone or in combination, also showed a tendency to progress towards hypertrophy, as judged by the expression of alkaline phosphatase and stainings for collagen type X and Annexin 5. CONCLUSION: As this study provides evidence for chondrogenic induction of MSC aggregates in vitro via IHH gene delivery, this technology may be efficiently employed for generating cartilaginous repair tissues in vivo.
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Condrogénesis/fisiología , Técnicas de Transferencia de Gen , Proteínas Hedgehog/genética , Células Madre Mesenquimatosas/fisiología , Diferenciación Celular/fisiología , Células Cultivadas , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: The appropriate tropism of adeno-associated virus (AAV) vectors that are systemically injected is crucial for successful gene therapy when local injection is not practical. Acidic oligopeptides have been shown to enhance drug delivery to bones. METHODS: In this study six-L aspartic acids (D6) were inserted into the AAV2 capsid protein sequence between amino acid residues 587 and 588. 129SVE mice were injected with double-stranded wild-type- (WT-) or D6-AAV2 mCherry expression vectors (3.24 x 1010 vg per animal) via the superficial temporal vein within 24 hours of birth. RESULTS: Fluorescence microscopy and quantitative polymerase chain reaction confirmed higher levels of mCherry expression in the paraspinal and gluteus muscles in the D6-AAV2 injected mice. The results revealed that although D6-AAV2 was less efficient in the transduction of immortalized cells stronger mCherry signals were detected over the spine and pelvis by live imaging in the D6-AAV2-injected mice than were detected in the WT-AAV2-injected mice. In addition, D6-AAV2 lost the liver tropism observed for WT-AAV2. CONCLUSIONS: An acidic oligopeptide displayed on AAV2 improves axial muscle tropism and decreases liver tropism after systemic delivery. This modification should be useful in creating AAV vectors that are suitable for gene therapy for diseases involving the proximal muscles.
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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. Appreciating the now apparent functional heterogeneity of osteosarcoma cells within and between individual tumors will likely be critical in developing much needed novel effective therapies. The foundation for this heterogeneity may lie in the so called "cancer stem cell" or tumorigenic cell of origin. In this brief review, we will examine the evidence for the existence of this cell and its potential importance for future therapies.
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Gene transfer technologies enable the controlled, targeted and sustained expression of gene products at precise anatomical locations, such as the joint. In this way, they offer the potential for more-effective, less-expensive treatments of joint diseases with fewer extra-articular adverse effects. A large body of preclinical data confirms the utility of intra-articular gene therapy in animal models of rheumatoid arthritis and osteoarthritis. However, relatively few clinical trials have been conducted, only one of which has completed phase II. This article summarizes the status in 2010 of the clinical development of gene therapy for arthritis, identifies certain constraints to progress and suggests possible solutions.
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Artritis Reumatoide/terapia , Terapia Genética/métodos , Osteoartritis/terapia , Reumatología/métodos , Artritis Reumatoide/genética , Ensayos Clínicos como Asunto , Técnicas de Transferencia de Gen , Humanos , Osteoartritis/genéticaRESUMEN
To understand the cellular and molecular events contributing to arthrofibrosis, we used an adenovirus to deliver and overexpress transforming growth factor-beta 1 (TGF-ß1) cDNA (Ad.TGF-ß1) in the knee joints of immunocompromised rats. Following delivery, animals were killed periodically, and joint tissues were examined macroscopically and histologically. PCR-array was used to assay alterations in expression patterns of extracellular matrix (ECM)-associated genes. By days 5 and 10, TGF-ß1 induced an increase in knee diameter and complete encasement of joints in dense scar-like tissue, locking joints at 90° of flexion. Histologically, massive proliferation of synovial fibroblasts was seen, followed by their differentiation into myofibroblasts. The fibrotic tissue displaced the normal architecture of the joint capsule and fused with articular cartilage. RNA expression profiles showed high levels of transcription of numerous MMPs, matricellular and ECM proteins. By day 30, the phenotype of the fibrotic tissue had undergone chondrometaplasia, indicated by cellular morphology, matrix composition and >100-fold increases in expression of collagen type II and cartilage link protein. Pre-labeling of articular cells by injection with recombinant lentivirus containing eGFP cDNA showed fibrotic/cartilaginous tissues appeared to arise almost entirely from local proliferation and differentiation of resident fibroblasts. Altogether, these results indicate that TGF-ß1 is a potent inducer of arthrofibrosis, and illustrate the proliferative potential and plasticity of articular fibroblasts. They suggest the mechanisms causing arthrofibrosis share many aspects with tumorigenesis.
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Condromatosis Sinovial/etiología , Articulaciones/patología , Factor de Crecimiento Transformador beta1/fisiología , Adenoviridae/genética , Animales , Cadherinas/genética , Factor de Crecimiento del Tejido Conjuntivo/genética , Fibroblastos/fisiología , Fibrosis , Perfilación de la Expresión Génica , Masculino , Metaloproteinasas de la Matriz/genética , Ratas , Ratas Desnudas , Ratas Wistar , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
INTRODUCTION: The present study compares bone morphogenetic protein (BMP)-4 and BMP-2 gene transfer as agents of chondrogenesis and hypertrophy in human primary mesenchymal stem cells (MSCs) maintained as pellet cultures. METHODS: Adenoviral vectors carrying cDNA encoding human BMP-4 (Ad.BMP-4) were constructed by cre-lox combination and compared to previously generated adenoviral vectors for BMP-2 (Ad.BMP-2), green fluorescent protein (Ad.GFP), or firefly luciferase (Ad.Luc). Cultures of human bone-marrow derived MSCs were infected with 5 x 10(2) viral particles/cell of Ad.BMP-2, or Ad.BMP-4, seeded into aggregates and cultured for three weeks in a defined, serum-free medium. Untransduced cells or cultures transduced with marker genes served as controls. Expression of BMP-2 and BMP-4 was determined by ELISA, and aggregates were analyzed histologically, immunohistochemically, biochemically and by RT-PCR for chondrogenesis and hypertrophy. RESULTS: Levels of BMP-2 and BMP-4 in the media were initially 30 to 60 ng/mL and declined thereafter. BMP-4 and BMP-2 genes were equipotent inducers of chondrogenesis in primary MSCs as judged by lacuna formation, strong staining for proteoglycans and collagen type II, increased levels of GAG synthesis, and expression of mRNAs associated with the chondrocyte phenotype. However, BMP-4 modified aggregates showed a lower tendency to progress towards hypertrophy, as judged by expression of alkaline phosphatase, annexin 5, immunohistochemical staining for type X collagen protein, and lacunar size. CONCLUSIONS: BMP-2 and BMP-4 were equally effective in provoking chondrogenesis by primary human MSCs in pellet culture. However, chondrogenesis triggered by BMP-2 and BMP-4 gene transfer showed considerable evidence of hypertrophic differentiation, with, the cells resembling growth plate chondrocytes both morphologically and functionally. This suggests caution when using these candidate genes in cartilage repair.
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Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 4/genética , Diferenciación Celular/genética , Condrocitos/citología , Condrogénesis/genética , Apoptosis/fisiología , Proteína Morfogenética Ósea 2/metabolismo , Proteína Morfogenética Ósea 4/metabolismo , Condrocitos/metabolismo , Técnicas de Transferencia de Gen , Humanos , Hipertrofia , Inmunohistoquímica , Técnicas In Vitro , Células Madre Mesenquimatosas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción Genética , TransgenesRESUMEN
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. In culture, only approximately 24% of the OS521Oct-4p cells were capable of activating the transgenic Oct-4 promoter; yet, xenograft tumors generated in NOD/SCID mice contained approximately 67% GFP(+) cells, which selectively expressed the mesenchymal stem cell-associated surface antigens CD105 and ICAM-1. Comparison of the tumor-forming capacity of GFP-enriched and GFP-depleted cell fractions revealed that the GFP-enriched fractions were at least 100-fold more tumorigenic, capable of forming tumors at doses of <300 cells, and formed metastases in the lung. Clonal populations derived from a single Oct-4/GFP(+) cell were capable of forming tumors heterogeneous for Oct-4/GFP expression. These data are consistent with the cancer stem cell model of tumorigenesis in osteosarcoma and implicate a functional link between the capacity to activate an exogenous Oct-4 promoter and tumor formation. This osteosarcoma tumor-initiating cell appears highly prolific and constitutes a majority of the cell population in a primary xenograft tumor, which may provide a biological basis for the particular virulence of this type of cancer.
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Células Madre Neoplásicas/patología , Factor 3 de Transcripción de Unión a Octámeros/genética , Osteosarcoma/patología , Regiones Promotoras Genéticas/genética , Animales , Antígenos CD/análisis , Línea Celular Tumoral , Endoglina , Femenino , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunohistoquímica , Molécula 1 de Adhesión Intercelular/análisis , Ratones , Ratones Endogámicos NOD , Ratones SCID , Microscopía Fluorescente , Trasplante de Neoplasias , Células Madre Neoplásicas/metabolismo , Osteosarcoma/genética , Osteosarcoma/metabolismo , Receptores de Superficie Celular/análisis , Sarcoma Experimental/genética , Sarcoma Experimental/metabolismo , Sarcoma Experimental/patología , Factores de Tiempo , TransfecciónRESUMEN
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. METHODS: Conventional and scAAV vectors were used to infect lapine articular fibroblasts in culture to determine transduction efficiency, transgene expression levels, and nuclear trafficking. scAAV containing the cDNA for interleukin (IL)-1 receptor antagonist (Ra) was delivered to the joints of naïve rabbits and those with IL-1beta-induced arthritis. From lavage of the joint space, levels of transgenic expression and persistence were measured by enzyme-linked immunosorbent assay. Infiltrating leukocytes were quantified using a hemocytometer. RESULTS: Transgene expression from scAAV had an earlier onset and was approximately 25-fold greater than conventional AAV despite the presence of similar numbers of viral genomes in the nuclei of infected cells. Fibroblasts transduced with scAAV produced amounts of IL1-Ra comparable to those transduced with adenoviral and lentiviral vectors. IL1-Ra was present in lavage fluid of most animals for 2 weeks in sufficient quantities to inhibit inflammation of the IL-1beta-driven model. Once lost, neither subsequent inflammatory events, nor re-administration of the virus could re-establish transgene expression. CONCLUSIONS: scAAV-mediated intra-articular gene transfer is robust and similarly efficient in both normal and inflamed joints; the resulting transgenic expression is sufficient to achieve biological relevance in joints of human proportion.