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Biomedical subjects

J Huard

Publications and source records attributed to J Huard.

At least 55 records · Page 3Linked to original sources
PubMed2000

Myoblast therapy for stress urinary incontinence and bladder dysfunction.

The field of tissue engineering and gene therapy has an exciting and promising future. During the past few years we have begun a comprehensive effort to investigate the use of myoblasts to improve and expand the treatment of stress urinary incontinence and bladder dysfunction. Moreover, we can expect the application of myoblast-mediated ex vivo gene transfer in the field of urology. In this paper we discuss the compositions of and methods involving the use of myogenic or muscle-derived cells for tissue engineering and cell-mediated gene therapy.

Cell Transplantation↗
PubMed2000

Matching host muscle and donor myoblasts for myosin heavy chain improves myoblast transfer therapy.

Intensive efforts have been made to develop an effective therapy for Duchenne muscular dystrophy (DMD). Although myoblast transplantation has been found capable of transiently delivering dystrophin and improving the strength of the injected dystrophic muscle, this approach has been hindered by the immune rejection problems as well as the poor survival and limited spread of the injected cells. In the present study, we have investigated whether the careful selection of donor myoblasts and host muscle for the myosin heavy chain expression (MyHCs) plays a role in the success of myoblast transfer. Highly purified normal myoblasts derived from the m. soleus and m. gastrocnemius white of normal mice were transplanted into the m. soleus (containing 70% of type I fibers) and gastrocnemius white (100% of type II fibers) of dystrophin deficient mdx mice. At several time-points after injection (10, 20 and 30 days), the number of dystrophin-positive fibers was monitored and compared among the different groups. A significantly higher number and better persistence of dystrophin-positive myofibers were observed when the injected muscle and donor myoblasts expressed a similar MyHC in comparison with myoblast transfer between host muscle and donor myoblasts that were not matched for MyHC. These results suggest that careful matching between the injected myoblasts and injected muscle for the MyHC expression can improve the efficiency of myoblast-mediated gene transfer to skeletal muscle. Gene Therapy (2000) 7, 428-437.

Animals↗
PubMed2000

Use of growth factors to improve muscle healing after strain injury.

Muscle injuries represent a large number of professional and recreational sports injuries. Muscle strains habitually occur after an eccentric contraction, which often leads to an injury located in the myotendinous junction. Treatment varies widely, depending on the severity of the trauma, but has remained limited mostly to rest, ice, compression, elevation, antiinflammatory drugs, and mobilization. The authors' research group aims to develop new biologic approaches to improve muscle healing after injuries, including muscle strains. To achieve this goal, the authors investigated several parameters that will lead to the development of new strategies to enhance muscle healing. The authors first evaluated natural muscle healing after strain injuries and showed that muscle regeneration occurs in the early phase of healing but becomes impaired with time by the development of tissue fibrosis. Several growth factors capable of improving muscle regeneration were investigated; basic fibroblast growth factor, insulin-like growth factor, and nerve growth factors were identified as substances capable of enhancing muscle regeneration and improving muscle force in the strained injured muscle. The current study should aid in the development of strategies to promote efficient muscle healing and complete recovery after strain injury.

Animals↗
PubMed2000

Ex vivo gene therapy to produce bone using different cell types.

Gene therapy and tissue engineering promise to revolutionize orthopaedic surgery. This study comprehensively compares five different cell types in ex vivo gene therapy to produce bone. The cell types include a bone marrow stromal cell line, primary muscle derived cells, primary bone marrow stromal cells, primary articular chondrocytes, and primary fibroblasts. After transduction by an adenovirus encoding for bone morphogenetic protein-2, all of the cell types were capable of secreting bone morphogenetic protein-2. However, the bone marrow stromal cell line and muscle derived cells showed more responsiveness to recombinant human bone morphogenetic protein-2 than did the other cell types. In vivo injection of each of the cell populations transduced to secrete bone morphogenetic protein-2 resulted in bone formation. Radiographic and histologic analyses corroborated the in vitro data regarding bone morphogenetic protein-2 secretion and cellular osteocompetence. This study showed the feasibility of using primary bone marrow stromal cells, primary muscle derived cells, primary articular chondrocytes, primary fibroblasts, and an osteogenesis imperfecta stromal cell line in ex vivo gene therapy to produce bone. The study also showed the advantages and disadvantages inherent in using each cell type.

Adenoviridae↗
PubMed2000

Growth factors improve muscle healing in vivo.

Injury to muscles is very common. We have previously observed that basic fibroblast growth factor (b-FGF), insulin growth factor type 1 (IGF-1) and nerve growth factor (NGF) are potent stimulators of the proliferation and fusion of myoblasts in vitro. We therefore injected these growth factors into mice with lacerations of the gastrocnemius muscle. The muscle regeneration was evaluated at one week by histological staining and quantitative histology. Muscle healing was assessed histologically and the contractile properties were measured one month after injury. Our findings showed that b-FGF, IGF and to a less extent NGF enhanced muscle regeneration in vivo compared with control muscle. At one month, muscles treated with IGF-1 and b-FGF showed improved healing and significantly increased fast-twitch and tetanus strengths. Our results suggest that b-FGF and IGF-1 stimulated muscle healing and may have a considerable effect on the treatment of muscle injuries.

Animals↗
PubMed1999

Adenovirus-mediated direct gene therapy with bone morphogenetic protein-2 produces bone.

The need to improve bone healing permeates the discipline of orthopedic surgery. Bone morphogenetic proteins (BMPs) are capable of inducing ectopic and orthotopic bone formation. However, the ideal approach with which to deliver BMPs remains unknown. Gene therapy to deliver BMPs offers several theoretical advantages over implantation of a recombinant BMP protein, including persistent BMP delivery and eliminating the need for a foreign body carrier. A replication defective adenoviral vector was constructed to carry the rhBMP-2 gene (AdBMP-2). The direct in vivo gene therapy approach was applied in both immunodeficient and immunocompetent animals to produce intramuscular bone as early as 2 weeks following injection. Radiographic and histologic analysis revealed radiodense bone containing mature bone marrow elements. Adenovirus-mediated delivery of a marker gene (beta-galactosidase) into control animals produced no bone but indicated the cells transduced with the AdBMP-2 vector. Furthermore, comparisons between immunodeficient and immunocompetent animals illustrated the magnitude and significance of the immune response. Gene therapy to deliver BMP-2 has innumerable potential clinical applications from bone defect healing to joint replacement prosthesis stabilization. This study is the first to establish the feasibility of in vivo gene therapy to deliver active BMP-2 and produce bone.

Adenoviridae↗
PubMed1999

Use of muscle cells to mediate gene transfer to the bone defect.

Segmental bone defects and nonunions are relatively common problems facing all orthopaedic surgeons. Osteogenic proteins, i.e., BMP-2, can promote bone healing in segmental bone defects. However, a large quantity of the human recombinant protein is needed to enhance the bone healing potential. Cell mediated gene therapy in the bone defect can allow a sustained expression of the osteogenic proteins and further enhance bone healing. Muscle cells can be easily isolated and cultivated, and they are known to be an efficient gene delivery vehicle to muscle and nonmuscle tissues. Furthermore, they are capable of transforming into osteoblasts when stimulated by BMP-2. Thus, the utilization of muscle cells as the gene delivery vehicle to a bone defect would be an important step in establishing a less invasive treatment for non-unions and segmental bone defects. Muscle cells were transduced when the adenoviral-lacZ vector and injected into the bone defect and the muscles surrounding the defect. Expression of the marker gene was visualized 7 days after the injection, both macroscopically and microscopically, using lacZ histochemistry. The lacZ expressing cells in the defect tissue were also stained for desmin, a muscle specific marker, indicating the presence of muscle cells that have fused into myofibers in this nonmuscle bone defect area. With successful myoblast mediated gene delivery into the segmental bone defect, future experiments would focus on delivering viral vectors expressing osteogenic proteins to eventually improve bone healing postinjury.

Animals↗
PubMed1999

Direct-, fibroblast- and myoblast-mediated gene transfer to the anterior cruciate ligament.

The anterior cruciate ligament (ACL) has poor capabilities of healing. Maturation or "ligamentization" of the ACL following autograft or allograft reconstruction has been found slow and remains under investigation. In vitro and in vivo studies have shown that platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta), and epidermal growth factor (EGF) have the potential to improve ligament healing. Gene therapy approaches may represent a new alternative in delivering these specific growth factors to the ACL. The aim of this study was to investigate the feasibility of three different gene therapy approaches (direct-, fibroblast-, and myoblast-mediated gene transfer) to the ACL. Rabbit myoblasts and ACL-fibroblasts were transduced with 5 x 10(7) recombinant adenoviral particles carrying the LacZ reporter gene (MOI = 50). Myoblasts and fibroblasts (1 x 10(6)) were each injected into the right ACL of 10 adult rabbits; direct injection of 5 x 10(7) adenoviral particles was performed in 10 other rabbits. The left side was used as sham. The beta-galactosidase production was revealed using the LacZ histochemical technique. The transduced fibroblasts and myoblasts were found in the ligament tissue and in the synovial tissue surrounding the ACL at 4, 7, 14, and 21 days postinjection. The myoblasts fused and formed myotubes in the ligament. The direct approach also allowed the transfer of the marker gene in the ligament at 4, 7, 21, and 42 days postinjection. X-gal staining revealed no expression of beta-galactosidase in the sham ligament. The presence of cells expressing the marker gene in the ACL opens up the possibility of delivering proteins (i.e., PDGF, TGF-beta, and EGF) capable of improving ACL healing and graft maturation. Furthermore, engineered myoblasts may mediate and accelerate the intraligament neovascularization. This new technology based on gene therapy and tissue engineering may allow a persistent expression of selected growth factors to enhance ACL healing following injury.

Adenoviridae↗
PubMed1999

The Pittsburgh Tissue Engineering Initiative.

The Pittsburgh Tissue Engineering Initiative (PTEI) is the prime catalyst for tissue-engineering research and the development of a viable for-profit tissue-engineering industry in southwestern Pennsylvania. PTEI operates targeted programs that support commercializable research; provide professional and community education; and facilitate interactions between academic medical centers, regional government, and industry to support the development of a vibrant tissue-engineering industry. This article discusses the types of research and education programs provided by PTEI and provides a brief summary of some of the tissue-engineering-related research in the region.

Biotechnology↗
PubMed1998

Development of approaches to improve cell survival in myoblast transfer therapy.

Myoblast transplantation has been extensively studied as a gene complementation approach for genetic diseases such as Duchenne Muscular Dystrophy. This approach has been found capable of delivering dystrophin, the product missing in Duchenne Muscular Dystrophy muscle, and leading to an increase of strength in the dystrophic muscle. This approach, however, has been hindered by numerous limitations, including immunological problems, and low spread and poor survival of the injected myoblasts. We have investigated whether antiinflammatory treatment and use of different populations of skeletal muscle-derived cells may circumvent the poor survival of the injected myoblasts after implantation. We have observed that different populations of muscle-derived cells can be isolated from skeletal muscle based on their desmin immunoreactivity and differentiation capacity. Moreover, these cells acted differently when injected into muscle: 95% of the injected cells in some populations died within 48 h, while others richer in desmin-positive cells survived entirely. Since pure myoblasts obtained from isolated myofibers and myoblast cell lines also displayed a poor survival rate of the injected cells, we have concluded that the differential survival of the populations of muscle-derived cells is not only attributable to their content in desmin-positive cells. We have observed that the origin of the myogenic cells may influence their survival in the injected muscle. Finally, we have observed that myoblasts genetically engineered to express an inhibitor of the inflammatory cytokine, IL-1, can improve the survival rate of the injected myoblasts. Our results suggest that selection of specific muscle-derived cell populations or the control of inflammation can be used as an approach to improve cell survival after both myoblast transplantation and the myoblast-mediated ex vivo gene transfer approach.

Animals↗
PubMed1998

Implications of maturation for viral gene delivery to skeletal muscle.

Different viral vectors have been analyzed as gene delivery vehicles to skeletal muscle for potentially therapeutic purposes. In this review, we evaluate the application of retroviral, adenoviral, and herpes simplex viral vectors to deliver genes to skeletal muscle and focus on the dramatic loss of viral transduction detected throughout muscle maturation. Recent results suggested that there are several factors involved in the reduced viral transducibility of mature skeletal muscle: muscle cells become post-mitotic in an early stage, the extracellular matrix develops into a physical barrier, and a loss of myoblast mediation occurs since myoblasts progressively become quiescent. Approaches to improve viral gene delivery to mature skeletal muscle may include the use of particular enzymes to increase the permeability of the extracellular matrix, the pre-treatment of the muscle with a myonecrotic agent to induce myoblast mediation, or the application of the myoblast-mediated ex vivo gene transfer.

Adenoviridae↗
PubMed1998

Muscle maturation: implications for gene therapy.

Skeletal muscle is a promising target tissue for gene therapy, for both muscle and non-muscle disorders. A variety of methods have been studied to transfer genes into skeletal muscle, including retroviral, adenoviral and herpes simplex viral vectors. However, various factors impede muscle-based viral gene therapy. Here, we discuss why some viral vectors cannot efficiently transduce mature muscle fibers, and describe some new approaches to overcome this barrier.

Animals↗
PubMed1998

Ex vivo gene transfer using adenovirus-mediated full-length dystrophin delivery to dystrophic muscles.

Duchenne muscular dystrophy (DMD) is an X-linked recessive muscle disease characterized by a lack of dystrophin expression. Myoblast transplantation and gene therapy have the potential of restoring dystrophin, thus decreasing the muscle weakness associated with this disease. In this study we present data on the myoblast mediated ex vivo gene transfer of full-length dystrophin to mdx (dystrophin deficient) mouse muscle as a model for autologous myoblast transfer. Both isogenic primary mdx myoblasts and an immortalized mdx cell line were transduced with an adenoviral vector that has all viral coding sequences deleted and encodes beta-galactosidase and full-length dystrophin. Subsequently, these transduced myoblasts were injected into dystrophic mdx muscle, where the injected cells restored dystrophin, as well as dystrophin-associated proteins. A greater amount of dystrophin replacement occurred in mdx muscle following transplantation of mdx myoblasts isolated from a transgenic mouse overexpressing dystrophin suggesting that engineering autologous myoblasts to express high amounts of dystrophin might be beneficial. The ex vivo approach possesses attributes that make it useful for gene transfer to skeletal muscle including: (1) creating a reservoir of myoblasts capable of regenerating and restoring dystrophin to dystrophic muscle; and (2) achieving a higher level of gene transfer to dystrophic muscle compared with adenovirus-mediated direct gene delivery. However, as observed in direct gene transfer studies, the ex vivo approach also triggers a cellular immune response which limits the duration of trans-gene expression.

Adenoviridae↗
PubMed1997

Viral gene delivery to skeletal muscle: insights on maturation-dependent loss of fiber infectivity for adenovirus and herpes simplex type 1 viral vectors.

The mechanisms causing age-dependent loss of muscle fiber infectivity observed in vivo for both adenoviral (Ad) and herpes simplex virus type 1 (HSV-1) gene delivery vectors remain poorly understood. Here we investigate the possible bases for this phenomenon using the novel application of enzymatically isolated, viable, single muscle fibers. We show that maturation-dependent loss of fiber infectivity is recapitulated in single fibers, and, thus, is not solely due to host immune response. Using localized irradiation of muscle in vivo, we show data suggesting that Ad infectivity of differentiated myofibers depends, at least in part, on myoblasts to mediate fiber transduction. On the other hand, infection of single fibers by HSV-1 is not affected by irradiation. Using confocal microscopy, we show that the basal lamina of myogenic cells efficiently infected by HSV-1 is structurally less organized than that of fibers resistant to infection by HSV-1. As well, we show that single myofibers isolated from adult, basal lamina-defective mice (merosin-deficient, dy/dy) are at least 10-fold more susceptible to infection by HSV-1 than are myofibers isolated from control mice. Together, these observations support the hypothesis that the basal lamina acts as a physical barrier to HSV-1 infection of mature muscle.

Adenoviridae↗
PubMed1997

LacZ gene transfer to skeletal muscle using a replication-defective herpes simplex virus type 1 mutant vector.

Herpes simplex virus type 1 (HSV-1) represents a promising new viral vector capable of efficient transduction of myofibers in vivo. Here we report on the use of a replication-defective HSV-1 mutant vector (DZ) deleted for the essential immediate early (IE) gene ICP4 for studies of reporter gene transfer and expression following direct inoculation of mouse skeletal muscle. The recombinant vector was engineered to contain the Escherichia coli lacZ gene under transcriptional control of the strong human cytomegalovirus (HCMV) IE promoter. The effect of vector cytotoxicity on the durability of transgene expression following infection of muscle cells in culture and myofibers in vivo revealed that this first-generation HSV vector was cytopathic, limiting the persistence of vector-transduced cells. UV irradiation of vector preparations reduced viral cytotoxicity for myoblasts in culture without reducing significantly beta-galactosidase production. Moreover, muscle cell viability and the durability of transgene expression was enhanced by several days following UV inactivated-vector infection in vivo. Nevertheless, the viral DNA was subsequently lost from vector-inoculated muscle tissue within 2 weeks. This observation indicated that vector toxicity alone did not account for the lack of persistent transgene expression. Longer-term vector transduction and transgene expression was observed, however, following inoculation of immunodeficient SCID mice, indicating that host immunocompetence played an important role in determining the duration of transgene expression in animals. To support this hypothesis, cells expressing CD4 and CD8 antigens have been found in the HSV-1 injected muscle of immunocompetent mice. These data demonstrated that both vector toxicity and vector-induced immunity are significant obstacles to the use of HSV-1 vectors for muscle gene transfer. These impediments must be overcome to further develop HSV vectors for muscle gene therapy applications.

Animals↗
PubMed1997

Limb lengthening promotes muscle growth.

Studies of limb lengthening have demonstrated successful bone formation in the distraction gap. Failure of the muscle units to lengthen leads to many complications that significantly limit the success of this approach; it is, therefore, of paramount importance to characterize the behavior of the muscle during limb lengthening. In this study, tibiae of adult rabbits were lengthened for 10 days at a rate of 1 mm/day. The proliferative ability of the lengthened muscle was characterized using bromodeoxyuridine, a thymidine analogue that is incorporated during cell division, and desmin, a muscle-specific marker. We observed a large number of proliferating cells, specifically in the lengthened muscle, that were co-localized with many desmin-positive cells. The presence of bromodeoxyuridine nuclei inside desmin-positive muscle fibers suggests that limb lengthening promotes muscle growth by triggering myoblast proliferation and fusion into the lengthened muscle. Our findings are consistent with those of other studies in the reviewed literature that also suggest that limb lengthening promotes muscle growth.

Animals↗
PubMed1997

Myoblast-mediated gene transfer to the joint.

Several genetic and acquired pathologic conditions of the musculoskeletal system, such as arthritis and damage to ligament, cartilage, and meniscus, may be amenable to gene therapy. Even though ex vivo gene transfer with synovial cells has been shown to deliver genes encoding for anti-arthritic proteins into the rabbit knee joint, its success has been limited by a transient transgene expression. In this study, data were investigated regarding the use of muscle cells as an alternative gene-delivery vehicle to the joint in newborn rabbit and adult severe combined immunodeficiency mice. We demonstrated that myoblasts were transduced more efficiently than synovial cells with use of the same adenoviral preparation in vitro. After intra-articular injection, the engineered muscle cells adhered to several structures in the joint, including the ligament, capsule, and synovium. In addition, myoblasts fused to form many post-mitotic myotubes and myofibers at different locations of the joint of the newborn rabbit 5 days after the injection. In the knee of the adult mouse, myoblasts fused and expressed the reporter gene for at least 35 days after the injection. The presence of post-mitotic myofibers in the knee joint raises the possibility of long-term expression of the secreted protein. Currently, numerous tissues in the joint (ligament, meniscus, and cartilage) have poor intrinsic healing capacity and frequently need surgical corrections. A stable gene-delivery vehicle to the joint producing proteins that ameliorate these different musculoskeletal conditions may change the clinical implications of these pathologies.

Adenoviridae↗