|Genes, scaffolds and cells for bone repair|
June 4, 2001
This week's feature highlights promising applications of gene and stem cell therapies for treating fractures and osteoporosis, along with the feasibility of whole bone marrow transplants for treating genetic diseases of the bone.
The reports discussed are points in a continuum of strategies being explored. Below is a collection of recent abstracts that describe other possible approaches. One report describes infusing a porous synthetic matrix with BMP-2 producing cells to stimulate bone formation. The advantage of using a scaffold is that it provides a template for bone formation.
Two abstracts address the question of targeting gene expression specifically to the bone-producing osteoblast cells. Each discusses a unique promoter that activates genes only in bone.
Finally, two abstracts report the delivery of IL-4 and IL-1 receptor antagonist proteins for the treatment of bone erosion in rheumatoid arthritis.
Bijal P. Trivedi
Currently, functional treatment of fracture non-unions and bone loss remains a significant challenge in the field of orthopaedic surgery. Tissue engineering of bone has emerged as a new treatment alternative in bone repair and regeneration. Our approach is to combine a polymeric matrix with a cellular vehicle for delivery of bone morphogenetic protein-2 (BMP-2), constructed through retroviral gene transfer. The objective of this study is to develop an osteoinductive, tissue-engineered bone replacement system by culturing BMP-2-producing cells on an osteoconductive, biodegradable, polymeric-ceramic matrix. The hypothesis is that retroviral gene transfer can be used effectively in combination with a biodegradable matrix to promote bone formation. First, we examined the in vitro attachment and growth of transfected BMP-producing cells on a PLAGA-HA scaffold. Second, the bioactivity of the produced BMP in vitro was evaluated using a mouse model. It was found that the polymer-ceramic scaffold supported BMP-2 production, allowing the attachment and growth of retroviral transfected, BMP-2-producing cells. In vivo, the scaffold successfully functioned as a delivery vehicle for bioactive BMP-2, as it induced heterotopic bone formation in a SCID mouse model.
Biomaterials 2001 Jun;22(11):1271-7.
Gene therapy of bone would benefit from the availability of vectors that provide stable, osteoblast-specific expression. This would allow bone-specific expression of Col1a1 cDNAs for treatment of osteogenesis imperfecta. In addition, such a vector would restrict expression of secreted therapeutic proteins to the bone-synthesizing regions of the bone marrow after ex vivo transduction of marrow stromal cells and reintroduction of the cells into patients. Retrovirus vectors stably integrate into target cell genomes; however, long-term regulated expression from internal cellular promoters has not been consistently achieved. In some cases this is due to a stem cell-specific mechanism for transcriptional repression of retroviruses. We evaluated the ability of self-inactivating ROSA-derived vectors containing a bone-directed 2.3-kb rat Col1a1 promoter to display osteoblast-specific expression. In vitro expression was examined in bone marrow stromal cell cultures induced to undergo osteoblastic differentiation. In vivo expression was evaluated in chimeric mice derived from transduced embryonic stem cells. The results indicate that self-inactivating retrovirus vectors containing the Col1a1 promoter are not permanently inactivated in embryonic stem cells and are specifically expressed in osteoblasts in vivo and in vitro. Thus these vectors should be useful for bone-directed gene therapy.
Mol Ther 2001 Apr;3(4):543-50.
Treatment of genetic or degenerative diseases severely affecting the entire skeleton may necessitate gene therapy involving transplantation of multipotential marrow cells. The ability of in vitro expanded adherent marrow cells enriched in pluripotent mesenchymal cell populations to remain competent to engraft, repopulate host tissues, and differentiate into bone and cartilage is advantageous for correction of skeletal-related diseases. However, to achieve phenotypic specificity and therapeutic or physiologic levels of proteins may require cell type specific expression of the gene. Tissue-specific promoter-controlled transgenes provide an efficacious approach to deliver therapeutic gene expression to repopulating chondrocytes and osteoblasts for treatment of cartilage and bone disorders or tumor metastasis to the skeleton. The bone-specific expression of a reporter gene controlled by the osteoblast-specific osteocalcin promoter after transplantation of a mixed population of marrow cells is shown. Tissue-restricted gene therapy potentially can be refined by use of a unique peptide targeting signal that directs the hematopoietic, chondrogenic, and osteogenic core binding factor/acute myelogenous leukemia transcription factors to subnuclear sites that support gene expression.
Clin Orthop 2000 Oct;(379 Suppl):S146-55.
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.
Clin Orthop 2000 Sep;(378):290-305.
Bone destruction is the most difficult target in the treatment of rheumatoid arthritis (RA). Here, we report that local overexpression of IL-4, introduced by a recombinant human type 5 adenovirus vector (Ad5E1mIL-4) prevents joint damage and bone erosion in the knees of mice with collagen arthritis (CIA). No difference was noted in the course of CIA in the injected knee joints between Ad5E1mIL-4 and the control vector, but radiographic analysis revealed impressive reduction of joint erosion and more compact bone structure in the Ad5E1mIL-4 group. Although severe inflammation persisted in treated mice, Ad5E1mIL-4 prevented bone erosion and diminished tartrate-resistant acid phosphatase (TRAP) activity, indicating that local IL-4 inhibits the formation of osteoclast-like cells. Messenger RNA levels of IL-17, IL-12, and cathepsin K in the synovial tissue were suppressed, as were IL-6 and IL-12 protein production. Osteoprotegerin ligand (OPGL) expression was markedly suppressed by local IL-4, but no loss of OPG expression was noted with Ad5E1mIL-4 treatment. Finally, in in vitro studies, bone samples of patients with arthritis revealed consistent suppression by IL-4 of type I collagen breakdown. IL-4 also enhanced synthesis of type I procollagen, suggesting that it promoted tissue repair. These findings may have significant implications for the prevention of bone erosion in arthritis.
J Clin Invest 2000 Jun;105(12):1697-710.
Gene therapy offers new possibilities for the clinical management of orthopaedic conditions that are difficult to treat by traditional surgical or medical means. To bring the potential of this novel technology into the clinic, a research program was initiated that aimed to identify orthopaedically useful genes and develop methods for delivering them to suitable sites under conditions in which gene expression remains at therapeutic levels for the appropriate periods of time; this program is now 10 years old. Rheumatoid arthritis was selected as the lead disease. Preclinical studies evaluating the local and systemic delivery of numerous different genes by in vivo and ex vivo methods in murine and lapin models led to the development of a human gene therapy protocol for arthritis. In this protocol, a gene encoding the human interleukin-1 receptor antagonist protein is transferred to the metacarpophalangeal joints of female patients with rheumatoid arthritis. The first patient was treated this way in July 1996. This is not only the first orthopaedic application of human gene therapy, but also the first use of gene therapy approved for a nonlethal disease. In addition to providing additional therapeutic options for the treatment of rheumatoid arthritis, the experimental data from this study suggest that gene transfer approaches may improve the treatment of osteoarthritis, the repair of cartilage, ligaments, tendons, menisci, intervertebral discs and bone, and the management of disorders such as osteoporosis and osteogenesis imperfecta. They also show promise as a means for developing novel and improved animal models of orthopaedic diseases. If the current rate of progress continues, wide clinical application of gene therapy in various orthopaedic indications should occur within the next 5 to 10 years.
Clin Orthop 2000 Jun;(375):324-37.
Bone morphogenetic proteins (BMPs) are capable of inducing endochondral bone formation when applied on biologic carriers in numerous mammalian in vivo assay systems. Bone morphogenetic protein gene therapy is also currently being developed to promote osteogenesis for clinical indications such as spinal fusions, craniofacial bone loss, and osteoporosis. In this study, critical-sized mandibular defects were treated with a control adenoviral vector (Ad-beta-gal), a BMP-2 adenoviral vector (Ad-BMP-2), or a BMP-9 adenoviral vector (Ad-BMP-9). Gross tissue examination, radiographic analysis, and histologic analysis demonstrated significant bony healing in the BMP treated groups compared to controls. Osteogenesis was limited to the bony defect, without extension into the surrounding soft tissues. The study suggests that with further development, BMP gene therapy may be potentially useful for repair of bony defects in the craniofacial region.
J Craniofac Surg 2000 Jan;11(1):24-30.
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