Axel W. A. Baltzer

Genetic enhancement of fracture repair: Healing of an experimental segmental defect by adenoviral transfer of the BMP-2 gene

This study evaluated the ability of gene transfer to enhance bone healing. Segmental defects were created surgically in the femora of New Zealand white rabbits. First generation adenoviruses were used as vectors to introduce into the defects genes encoding either human bone morphogenetic protein-2 (BMP-2) or, as a negative control, firefly luciferase. Representative specimens were evaluated histologically after 8 weeks. Healing of the defects was monitored radiographically for 12 weeks, after which time the repair tissue was evaluated biomechanically. By radiological criteria, animals receiving the BMP-2 gene had healed their osseous lesions after 7 weeks, whereas those receiving the luciferase gene had not. Histologic examination of representative rabbits at 8 weeks confirmed ossification across the entire defect in response to the BMP-2 gene, whereas the control defect was predominantly fibrotic and sparsely ossified. At the end of the 12-week experiment, the control femora still showed no radiological signs of stable healing. The difference in radiologically defined healing between the experimental and control groups was statistically significant (P < 0.002). biomechanical testing of the femora at 12 weeks demonstrated statistically significant increases in the mean bending strength (p < 0.005) and bending stiffness (p < 0.05) of the animals treated with the bmp-2 gene. direct, local adenoviral delivery of an osteogenic gene thus led to the healing of an osseous lesion that otherwise would not do so. these promising data encourage the further development of genetic approaches to enhancing bone healing.

Effect of BMP-2 gene transfer on bone healing in sheep

Critical size defects of bone and delayed fracture healing due to metabolic disorders are still problems in orthopaedic surgery. Adenoviral vectors encoding bone morphogenetic protein-2 (Ad.BMP-2) have been used to stimulate bone formation in small animals. The present study evaluated the use of direct adenoviral gene transfer for inducing bone formation in a large animal. Standardized iliac crest defects were created surgically on both sides of the pelvic bone of white mountain sheep. The efficiency of gene transfer was evaluated using recombinant adenoviruses carrying the cDNA for luciferase. High levels of transgene expression, restricted to the site of injection, were found for the 1st week. Transgene expression then fell considerably, but could still be detected for up to 5 weeks. To investigate the effect on bone healing, Ad.BMP-2 (1011 particles in 200 μl saline) was unilaterally injected into iliac crest defects and into tibial osteotomies. The contralateral defects remained untreated to evaluate possible systemic effects. The controls were treated with saline solution. Bone formation within the defect, assessed by micro-computed tomography (CT) measurement at 8 weeks, and callus formation after osteotomy were significantly reduced following direct application of Ad.BMP-2. The retardation compared to untreated control animals was additionally found at the contralateral iliac crest indicating a systemic inhibitory effect. Histological analysis confirmed the CT measurement and showed an increased number of inflammatory cells within both defects. Antibodies against the adenovirus and the transgene product were detected in all treated animals. These data show a systemic retardation of bone formation following a single local injection of Ad.BMP-2 in sheep. This finding stands in contrast to the data obtained from small animal models. Further studies are needed to determine the contribution of the immune response to these results, and whether a lower dose of Ad.BMP-2 would be advantageous.

A gene therapy approach to accelerating bone healing Evaluation of gene expression in a New Zealand white rabbit model

It has been demonstrated that BMPs, IGFs, and TGFβs improve the process of bone healing in vivo. We have suggested the use of gene therapy as a possible way to deliver growth factors to fracture sites in order to improve repair. The aim of this study was to develop a minimally invasive gene therapy approach to treat bone injuries locally without damaging the local blood circulation. A segmental defect of 1.3 cm was created in the diaphysis of the femur in mature NZW rabbits. Internal fixation with 7-hole DCP plates and 2.7 mm screws was used to stabilize the bone. After building a chamber by tightly closing the muscles around the segmental defect, 0.5 ml of either saline solution or a collagen gel containing 1 × 1010 particles of adenovirus carrying cDNA encoding either the bacterial β-galactosidase gene (LacZ), or the firefly luciferase gene were injected into the gap. The control side received 0.5 ml of saline solution without virus particles. Bone marrow, cortical and trabecular bone and surrounding muscle were harvested from the injected femur and were analyzed for local gene expression through X-gal staining or measurement of local luciferase activity. To determine whether distant sites were transduced, tissue from the spleen, liver, and lung were harvested as well as bone, bone marrow and muscle from the contralateral diaphysis of the femur. The delivery of the adenoviral vector suspended in saline solution led to local transduction of the bone, bone marrow and the muscle surrounding the gap. No luciferase activity was found in the contralateral femur, lung, or spleen, and only transient luciferase activity was seen in the liver. While marker gene expression persisted within the surrounding soft tissues for at least 2 weeks, the expression in bone lasted up to 6 weeks. This study has shown that it is possible to use adenoviral vectors to transfer and express genes locally within a segmental defect. Gene expression persisted for several weeks, which may be already sufficient to accelerate repair.

Potential role for gene therapy in the enhancement of fracture healing

Various proteins have the potential to initiate and accelerate fracture healing. Although osteogenic growth factors are the most prominent of these, there also may be important roles for other agents including growth factor receptors, angiogenic factors, and cytokine antagonists. Gene based delivery systems offer the potential to achieve therapeutic levels of these proteins locally within the fracture site for sustained times. Moreover, these delivery systems may deliver their products in a more biologically active form than that achieved by the exogenous application of recombinant proteins. Genes may be transferred to fractures by direct in vivo delivery or by indirect ex vivo delivery, using viral or nonviral vectors. Two examples are described in this article. With an ex vivo procedure, it was possible to transfer lac Z and neo(r) marker genes to the bones of mice, using retroviral transduction of bone marrow stromal cells. Gene expression in vivo persisted for several weeks. This procedure has the advantage of providing not only gene products but also osteoprogenitor cells to sites of bone healing. In vivo, local transfer of the lucerifase and lac Z marker genes was accomplished in a segmental defect model in the rabbit using adenoviral vectors. Under these conditions, gene expression in most tissues in and around the defect lasted between 2 and 6 weeks. These data encourage additional development of gene therapy for fracture healing. Such developments should go hand in hand with studies in the basic biology of fracture healing.

Potential role of direct adenoviral gene transfer in enhancing fracture repair.

Gene therapy has much to offer in the treatment of conditions in which it is necessary to increase the formation of bone. Nonunions, segmental defects, and aseptic loosening are examples of conditions where the local expression of genes that inhibit osteolysis and promote osteogenesis might be helpful. Studies in which one such possibility has been evaluated experimentally are described. These investigations used a surgically produced segmental defect in the femurs of New Zealand White rabbits as the model system. Adjacent muscle was fashioned around the defect to form a chamber into which adenoviral vectors were injected. High levels of transgene expression were found in the muscle surrounding the defect after injection of vectors carrying marker genes. Transgene expression also was seen in the cut ends of the bone and the scar tissue within the gap. No transgene expression was seen in the contralateral limb, spleen, or lung; transient, low levels of expression were found in the liver. Transgene expression declined with time, disappearing from all tissue but bone by Day 26; expression persisted in bone for at least 6 weeks. The control defects did not heal spontaneously. Injection of adenovirus carrying a human bone morphogenetic protein-2 complementary deoxyribonucleic acid led to healing of the segmental defect within 12 weeks, as judged by radiographic, histologic, and biomechanical criteria. Adenovirus carrying a human transforming growth factor-beta 1 complementary deoxyribonucleic acid showed signs of improved healing, but not to the extent seen with the bone morphogenetic protein-2 complementary deoxyribonucleic acid. This approach to therapy holds much promise as a novel means of promoting osteogenesis.

Clinical Responses to Gene Therapy in Joints of Two Subjects with Rheumatoid Arthritis

This paper provides the first evidence of a clinical response to gene therapy in human arthritis. Two subjects with rheumatoid arthritis received ex vivo, intraarticular delivery of human interleukin-1 receptor antagonist (IL-1Ra) cDNA. To achieve this, autologous synovial fibroblasts were transduced with a retrovirus, MFG-IRAP, carrying IL-1Ra as the transgene, or remained as untransduced controls. Symptomatic metacarpophalangeal (MCP) joints were injected with control or transduced cells. Joints were clinically evaluated on the basis of pain; the circumference of MCP joint 1 was also measured. After 4 weeks, joints underwent surgical synovectomy. There were no adverse events in either subject. The first subject responded dramatically to gene transfer, with a marked and rapid reduction in pain and swelling that lasted for the entire 4 weeks of the study. Remarkably, joints receiving IL-1Ra cDNA were protected from flares that occurred during the study period. Analysis of RNA recovered after synovectomy revealed enhanced expression of IL-1Ra and reduced expression of matrix metalloproteinase-3 and IL-1beta. The second subject also responded with reduced pain and swelling. Thus, gene transfer to human, rheumatoid joints can be accomplished safely to produce clinical benefit, at least in the short term. Using this ex vivo procedure, the transgene persisted within the joint for at least 1 month. Further clinical studies are warranted.

The potential of gene therapy for fracture healing in osteoporosis

Osteoporosis-associated fractures impair a patient’s function and quality of life and represent one of the major public health burdens. Demographic changes predict a dramatic increase in osteoporotic fractures. Experimental data have shown that osteoporosis impairs fracture healing. Clinical observations demonstrate high failure rates of implant fixation in osteoporosis. The reduced healing capacity, including impaired bone formation, in osteoporotic humans might be due to defects in mesenchymal stem cells that lead to reduced proliferation and osteoblastic differentiation. Growth factors show remarkable promise as agents that can improve the healing of bone or increase the proliferation and differentiation capacities of mesenchymal stem cells. Their clinical utility is limited by delivery problems. The attraction of gene-transfer approaches is the unique ability to deliver authentically processed gene products to precise anatomical locations at therapeutic levels for sustained periods of time. Unlike the treatment of chronic diseases, it is neither necessary nor desirable for transgene expression to persist beyond the few weeks or months needed to achieve healing. This review presents different approaches of gene therapy to enhance fracture healing and summarizes the promising results of preclinical studies. It focuses on applications of this new technique to fracture healing in osteoporosis. In our opinion, these applications represent some of the few examples in which gene therapy has a good chance of early clinical success.

Gene therapy for osteoporosis: evaluation in a murine ovariectomy model

Various cytokines and cytokine antagonists hold promise as new therapeutic agents for osteoporosis, but their application is hindered by delivery problems. Gene transfer offers an attractive technology with which to obviate these restrictions. Its utility was evaluated in an animal model of osteoporosis. Disease was induced by surgical ovariectomy and monitored by measuring bone weight after 12 days, and by histomorphometry after 5 weeks. Genes were transferred to the mice by intramedullary injection of adenoviral vectors. LacZ and luciferase marker genes were used to identify the bone marrow cells transduced by this procedure, and to track the possible spread of transgenes to other organs. The effect on bone loss of transferring a cDNA encoding the human interleukin-1 receptor antagonist (IL-1Ra) was then evaluated. The intramedullary injection of adenoviral vectors transduced lining osteoblasts, osteocytes and cells within the bone marrow. Luciferase activity persisted within the injected femora and adjacent musculature for at least 3 weeks, and in the draining lymph nodes for 2 weeks. Transient, low level expression was present in the liver, but no luciferase was detected at any time in the lung or spleen. Intramedullary introduction of the IL-1Ra gene resulted in circulation of the corresponding protein at concentrations that peaked on day 3, and returned to baseline by day 12. Transfer of the IL-1Ra gene strongly reduced the early loss of bone mass occurring in response to ovariectomy. Furthermore, it completely inhibited the loss of matrix detected by histomorphometry at 5 weeks. The protective effect of this gene was not restricted to bones receiving intramedullary injection of the vector, but occurred in all bones that were evaluated. This proof of concept encourages further development of gene therapy approaches to the treatment of osteoporosis.

American football injuries in Germany First results from Bundesliga football

Abstract This study investigated the incidence of injury in German Bundesliga football based on the evaluation of two American football teams before and during the season 1995. Certified team physicians and team physiotherapists were the initial medical professionals providing on-site diagnosis, injury documentation and first aid for all injuries. An injury was defined as minor (group I) causing the player to miss practice or game sessions up to 1 week without requiring surgery; as severe (group II) causing the player to miss practice or game sessions for more than 1 week up to 3 months or requiring surgery; and as catastrophic (group III) if the incident led to treatment for more than 3 months, treatment in an intensive care unit or persistent neurological or orthopaedic disability or death. From the data collected it was possible to calculate the risk of injury per time of exposure per athlete. The athlete’s function, influence of the weather and mechanisms of the injuries were registered. In total, 242 injuries were documented. The rate of injury was calculated as 16 per 1000 hours of practice and game per athlete. Severe injuries (group II) were found in 94 cases. Catastrophic injuries were not seen during the study. The knee was found to be the most common site of injury, while the ankle ranked second. Our study showed that the risk of injury in American football in German Bundesliga is comparable with soccer or handball.

A new treatment for hip osteoarthritis: clinical evidence for the efficacy of autologous conditioned serum

The purpose of the present study was to investigate the effect of intra-articular injections of autologous conditioned serum on human hip osteoarthritis and to test whether a potential treatment effect might be increased by additional injections of steroids and the recombinant interleukin-1 receptor antagonist protein anakinra. We compared the effects of autologous conditioned serum 46 hip osteoarthritis patients), autologous conditioned serum+cortisone (56 patients), and autologous conditioned serum+cortisone+recombinant interleukin-1 receptor antagonist protein (17 patients) in a retrospective clinical study by means of the Visual Analogue Scale for pain (pre- vs posttreatment). Over 14 months, treatment resulted in a large, statistically significant improvement for patients in all three groups, independent of the severity of osteoarthritis. Neither cortisone nor cortisone+recombinant interleukin-1 receptor antagonist protein increased the beneficial treatment effect over and above the effect of autologous conditioned serum alone. Autologous conditioned serum successfully reduces pain in hip osteoarthritis. In severe hip osteoarthritis, the sole application of autologous conditioned serum can be even more beneficial than the combination of autologous conditioned serum with steroids.