Effect of ACL reconstruction tunnels on stress in the distal femur.

PURPOSE: This study examined the change in femoral stress caused by graft tunnels drilled for anterior cruciate ligament (ACL) reconstruction. Using a computational model, the number, geometry and position of the graft tunnels exits were varied to determine the effect on bone stress. METHODS: A finite element model of the distal femur was developed from a CT scan of a cadaveric knee. To assess the model, the strain calculated computationally was compared to experimentally measured strains in eleven unpaired human cadaver femurs. Using the computational model, the number, geometry and position of the graft tunnel exits were varied to determine the effect on bone stress based on the stress concentration factor: the ratio of bone stress with tunnels to intact bone stress. RESULTS: The results indicated that the second tunnel in double-bundle ACL reconstruction results in approximately a 20 % increase in the maximum femoral stress as compared to single-bundle reconstruction. The highest stresses occur at the tunnel exits. The position of the tunnel exits effects femoral stress with the stress increasing slightly (AM SCR from 0.7 to 1 and PL SCR from 1.2 to 1.3) when the AM tunnel exit is moved anteriorly and having greater increases as the posterior lateral (PL) tunnel exit is moved laterally (PL SCR from 1.2 to 1.7) or posteriorly (PL SCR from 1.2 to 2). CONCLUSION: In anatomical ACL reconstruction, the tunnel entrances are dictated by anatomy; however, there can be variations in tunnel exit positions. Consideration should be given when positioning tunnel exits on the effect on stress in the femur. Moving the PL tunnel exit laterally or posteriorly increases in the stress at the PL tunnel exit.

Analysis of power law models for the creep of nucleus pulposus tissue.

Tissue engineering approaches are now being investigated for altering the course of intervertebral disc degeneration (IDD). Because the disease changes the mechanical properties of the load bearing tissues of the disc, viscoelastic tissue behavior is a key measure for comparing the efficacy of treatments. To investigate the basic viscoelastic behavior of nucleus pulposus tissue, tissue from the rabbit disc was tested in torsional creep. Both the Andrade and Nutting creep models had a good fit to the data, however, the Andrade creep model gave a much better prediction of the longer term creep. This is the first application of Andrade creep to biological tissue and results indicate that this model may be particularly well suited for characterizing the viscoelastic behavior of very soft biological tissues.

Individual patient data meta-analysis of randomized controlled trials of community occupational therapy for stroke patients.

BACKGROUND AND PURPOSE: Trials of occupational therapy for stroke patients living in the community have varied in their findings. It is unclear why these discrepancies have occurred. METHODS: Trials were identified from searches of the Cochrane Library and other sources. The primary outcome measure was the Nottingham Extended Activities of Daily Living (NEADL) score at the end of intervention. Secondary outcome measures included the Barthel Index or the Rivermead ADL (Personal ADL), General Health Questionnaire (GHQ), Nottingham Leisure Questionnaire (NLQ), and death. Data were analyzed using linear or logistic regression with a random effect for trial and adjustment for age, gender, baseline dependency, and method of follow-up. Subgroup analyses compared any occupational therapy intervention with control. RESULTS: We included 8 single-blind randomized controlled trials incorporating 1143 patients. Occupational therapy was associated with higher NEADL scores at the end of intervention (weighted mean difference [WMD], 1.30 points, 95% confidence intervals [CI], 0.47 to 2.13) and higher leisure scores at the end of intervention (WMD, 1.51 points; 95% CI, 0.24 to 2.79). Occupational therapy emphasizing activities of daily living (ADL) was associated with improved end of intervention NEADL (WMD, 1.61 points; 95% CI, 0.72 to 2.49) and personal activities of daily living (odds ratio [OR], 0.65; 95% CI, 0.46 to 0.91), but not NLQ. Leisure-based occupational therapy improved end of intervention NLQ (WMD, 1.96 points; 95% CI, 0.27 to 3.66) but not NEADL or PADL. CONCLUSIONS: Community occupational therapy significantly improved personal and extended activities of daily living and leisure activity in patients with stroke. Better outcomes were found with targeted interventions.

Gene therapy for degenerative disc disease.

Degenerative disc disease (DDD) is a chronic process that can become clinically manifest in multiple disorders such as idiopathic low back pain, disc herniation, radiculopathy, myelopathy, and spinal stenosis. The limited available technology for the treatment of these and other pathologic and disabling conditions arising from DDD is highly invasive (eg, surgical discectomy and fusion), manifesting a certain degree of complications and unsatisfactory clinical outcomes. Although the precise pathophysiology of DDD remains to be clearly delineated, the progressive decline in aggrecan, the primary proteoglycan of the nucleus pulposus, appears to be a final common pathway. It has been hypothesized that imbalance in the synthesis and catabolism of certain critical extracellular matrix components can be mitigated by the transfer of genes to intervertebral disc cells encoding factors that modulate synthesis and catabolism of these components. The successful in vivo transfer of therapeutic genes to target cells within the intervertebral disc in clinically relevant animal models of DDD is one example of the rapid progress that is being made towards the development of gene therapy approaches for the treatment of DDD. This chapter reviews the ability of gene therapy to alter biologic processes in the degenerated intervertebral disc and outlines the work needed to be done before human clinical trials can be contemplated.

Cre/lox-mediated marker gene excision in transgenic maize (Zea mays L.) plants.

After the initial transformation and tissue culture process is complete, selectable marker genes, which are used in virtually all transformation approaches, are not required for the expression of the gene of interest in the transgenic plants. There are several advantages to removing the selectable marker gene after it is no longer needed, such as enabling the reuse of selectable markers and simplifying transgene arrays. We have tested the Cre/ lox system from bacteriophage P1 for its ability to precisely excise stably integrated marker genes from chromosomes in transgenic maize plants. Two strategies, crossing and autoexcision, have been tested and demonstrated. In the crossing strategy, plants expressing the Cre recombinase are crossed with plants bearing a transgene construct in which the selectable marker gene is flanked by directly repeated lox sites. Unlike previous reports in which incomplete somatic and germline excision were common, in our experiments complete somatic and germline marker gene excision occurred in the F(1) plants from most crosses with multiple independent Cre and lox lines. In the autoexcision strategy, the cre gene, under the control of a heat shock-inducible promoter, is excised along with the nptII marker gene. Our results show that a transient heat shock treatment of primary transgenic callus is sufficient for inducing cre and excising the cre and nptII genes. Genetic segregation and molecular analysis confirmed that marker gene removal is precise, complete and stable. The autoexcision strategy provides a way of removing the selectable marker gene from callus or other tissues such as embryos and kernels.

Cross-linking to improve THR wear performance.

Polyethylene wear and associated osteolysis can limit the longevity of total hip replacement. In recent years, many improvements have been made in the consolidation, manufacture, and sterilization of polyethylene acetabular components. These improvements provided reduced polyethylene wear and prolonged usefulness of total hip replacement. Recent advances in extensively cross-linking polyethylene offer the possibility to substantially further reduce wear in total hip replacement. Hip simulator wear testing demonstrates an order of magnitude reduction in wear resulting from cross-linking GUR 1050 polyethylene by exposure to 100 kGy of electron beam radiation followed by annealing to encourage cross-linking and to reduce residual free radicals. Clinical investigation will be required to validate the wear advantage of these materials in vivo. (Hip International 2002; 2: 103-7).

New use of a three-dimensional pellet culture system for human intervertebral disc cells: initial characterization and potential use for tissue engineering.

STUDY DESIGN: Human intervertebral disc cells were cultured in a new three-dimensional "pellet culture" system as an alternative to conventional alginate bead microspheres. Histologic, biochemical, and immunohistologic assays were performed to characterize this new culturing method for disc cells. The feasibility of using the pellet culture system to study effects of gene therapy was also assessed. OBJECTIVES: To characterize a new and simpler, three-dimensional culture system for human intervertebral disc cells and to assess the feasibility of its use for gene therapy and tissue engineering studies. SUMMARY OF BACKGROUND DATA: The alginate microsphere three-dimensional culture system has been the most utilized culture method for disc cells, but it is technically difficult and has some disadvantages. Recently, the "pellet culture" method, a simpler three-dimensional culture system, was described for bone marrow stromal cells. This simpler method might be useful for in vitro and in vivo studies of disc cells as well as for delivery of exogenous genes. METHODS: Isolated human intervertebral disc cells were centrifuged at low speeds to form aggregates and allowed to grow as pellets for up to 3 weeks. At various times these pellet cultures were analyzed grossly, histologically, and immunohistologically. Their ability to incorporate [35S]sulfate in their response to TGF-beta1 was also analyzed. The ability of these pellets to deliver and express exogenous genes in vivo was analyzed by implantation of pellet cultures in muscles of SCID mice. RESULTS: Within several days the intervertebral disc cells were able to form mature three-dimensional aggregates that were each well encapsulated by a fibrous capsule. These pellets successfully synthesized proteoglycan and collagen Type II matrix as determined by histology and immunohistochemistry. In response to TGF-beta1, the pellets increased synthesis of proteoglycan and collagen Type II. When implanted into thigh muscles of SCID mice, the pellets remained aggregated and expressed the beta-galactosidase marker gene in vivo for up to 2 weeks. CONCLUSIONS: The pellet culture system is a new and technically simple method to culture human intervertebral disc cells. The majority of the human intervertebral disc cells retained their native phenotype in this three dimensional system and expressed a marker gene both in vitro and in vivo. Thus, this pellet system might be useful for in vitro and in vivo biochemical studies as well as for studies involving gene therapy and tissue engineering.

Multiple pathways for Cre/lox-mediated recombination in plastids.

Plastid transformation technology involves the insertion by homologous recombination and subsequent amplification of plastid transgenes to approximately 10 000 genome copies per leaf cell. Selection of transformed genomes is achieved using a selectable antibiotic resistance marker that has no subsequent role in the transformed line. We report here a feasibility study in the model plant tobacco, to test the heterologous Cre/lox recombination system for antibiotic marker gene removal from plastids. To study its efficiency, a green fluorescent protein reporter gene activation assay was utilized that allowed visual observation of marker excision after delivery of Cre to plastids. Using a combination of in vivo fluorescence activation and molecular assays, we show that transgene excision occurs completely from all plastid genomes early in plant development. Selectable marker-free transplastomic plants are obtained in the first seed generation, indicating a potential application of the Cre/lox system in plastid transformation technology. In addition to the predicted transgene excision event, two alternative pathways of Cre-mediated recombination were also observed. In one alternative pathway, the presence of Cre in plastids stimulated homologous recombination between a 117 bp transgene expression element and its cognate sequence in the plastid genome. The other alternative pathway uncovered a plastid genome 'hot spot' of recombination composed of multiple direct repeats of a 5 bp sequence motif, which recombined with lox independent of sequence homology. Both recombination pathways result in plastid genome deletions. However, the resultant plastid mutations are silent, and their study provides the first insights into tRNA accumulation and trans-splicing events in higher plant plastids.

Load-sharing between anterior and posterior elements in a lumbar motion segment implanted with an artificial disc.

STUDY DESIGN: A nonlinear three-dimensional finite element model of the osteoligamentous L3-L4 motion segment was used to predict changes in posterior element loads as a function of disc implantation and associated surgical procedures. OBJECTIVES: To evaluate the effects of disc implantation on the biomechanics of the posterior spinal elements (including the facet joints, pedicles, and lamina) and on the vertebral bodies. SUMMARY OF BACKGROUND DATA: Although several artificial disc designs have been used clinically, biomechanical data-particularly the change in loads in the posterior elements after disc implantation-are sparse. METHODS: A previously validated intact finite element model was implanted with a ball-and-cup-type artificial disc model via an anterior approach. The implanted model predictions were compared with in vitro data. To study surgical variables, small and large windows were cut into the anulus, and the implant was placed anteriorly and posteriorly within the disc space. The anterior longitudinal ligament was also restored. Models were subjected to either 800 N axial compression force alone or to a combination of 10 N-m flexion-extension moment and 400 N axial preload. Implanted model predictions were compared with those of the intact model. RESULTS: Facet loads were more sensitive to the anteroposterior location of the artificial disc than to the amount of anulus removed. Under 800 N axial compression, implanted models with an anteriorly placed artificial disc exhibited facet loads 2.5 times greater than loads observed with the intact model, whereas posteriorly implanted models predicted no facet loads in compression. Implanted models with a posteriorly placed disc exhibited greater flexibility than the intact and implanted models with anteriorly placed discs. Restoration of the anterior longitudinal ligament reduced pedicle stresses, facet loads, and extension rotation to nearly intact levels. CONCLUSIONS: The models suggest that, by altering placement of the artificial disc in the anteroposterior direction, a surgeon can modulate motion-segment flexuralstiffness and posterior load-sharing, even though the specific disc replacement design has no inherent rotational stiffness.

Human intervertebral disc cells are genetically modifiable by adenovirus-mediated gene transfer: implications for the clinical management of intervertebral disc disorders.

STUDY DESIGN: Human intervertebral disc cells were cultured in monolayer and treated with adenovirus-containing marker genes to determine the susceptibility of the cells to adenovirus-mediated gene transfer. OBJECTIVES: To test the efficacy of the adenovirus-mediated gene transfer technique for transferring exogenous genes to human intervertebral disc cells in vitro. SUMMARY OF BACKGROUND DATA: Upregulated proteoglycan synthesis after direct in vivo adenovirus-mediated transfer of growth factor genes to the rabbit intervertebral disc has previously been reported. Before contemplating extending this approach to the treatment of human disc disease, it is necessary to demonstrate that human intervertebral disc cells are indeed susceptible to adenovirus-mediated gene transduction. METHODS: Human intervertebral disc cells were isolated from disc tissue obtained from 15 patients during surgical disc procedures. The cells were cultured in monolayer and treated with saline containing five different doses of adenovirus carrying the lacZ gene (Ad/CMV-lacZ), saline containing adenovirus carrying the luciferase gene (Ad/CMV-luciferase), or saline alone. Transgene expression was analyzed by 5-bromo-4-chloro-3-indolyl-beta-galactosidase (X-Gal) staining and luciferase assay. RESULTS: Adenovirus efficiently transferred lacZ and luciferase marker genes to cells from degenerated discs as well as to cells from nondegenerated discs. A minimum dose of 150 MOI Ad/CMV-lacZ was found to be sufficient to achieve transduction of approximately 100% of disc cells-regardless of patient age, sex, surgical indication, disc level, and degeneration grade. No statistically significant difference in the luciferase activities could be detected in disc cell cultures from degenerated and nondegenerated discs treated with Ad/CMV-luciferase. CONCLUSIONS: In vitro transducibility of human intervertebral disc cells by adenovirus is relatively insensitive to disc degeneration grade. Because the rate-limiting step for successful gene therapy is the ability to transfer genes efficiently to the target tissue, the achievement of efficient gene transfer to human intervertebral disc cells(using a direct, adenovirus-mediated approach) is an important and necessary step in the development of gene therapy strategies for the management of human intervertebral disc disorders.

Potential applications of gene therapy to the treatment of intervertebral disc disorders.

Gene therapy involves the transfer of genes to cells such that the recipient cells express these genes and thereby synthesize the ribonucleic acid and protein that they encode. Recent investigations suggest that gene therapy may have potential applications in the treatment of intervertebral disc disorders, particularly those associated with disc degeneration. The successful in vivo transfer of therapeutic genes to target cells within the intervertebral disc in clinically relevant animal models is one example of the rapid progress that is being made. The purpose of the current review is to address several important technical issues, including choice of vectors and gene delivery strategy and the characteristics of the target tissues, which are relevant to future clinical applications of gene therapy for the treatment of intervertebral disc disorders. It already is apparent from the growing literature that gene therapy has the potential of becoming a valuable clinical treatment mode for intervertebral disc disorders in the twenty-first century.

Domiciliary occupational therapy for patients with stroke discharged from hospital: randomised controlled trial.

OBJECTIVE: To establish if a brief programme of domiciliary occupational therapy could improve the recovery of patients with stroke discharged from hospital. DESIGN: Single blind randomised controlled trial. SETTING: Two hospital sites within a UK teaching hospital. SUBJECTS: 138 patients with stroke with a definite plan for discharge home from hospital. INTERVENTION: Six week domiciliary occupational therapy or routine follow up. MAIN OUTCOME MEASURES: Nottingham extended activities of daily living score and "global outcome" (deterioration according to the Barthel activities of daily living index, or death). RESULTS: By eight weeks the mean Nottingham extended activities of daily living score in the intervention group was 4.8 points (95% confidence interval -0.5 to 10.0, P=0.08) greater than that of the control group. Overall, 16 (24%) intervention patients had a poor global outcome compared with 30 (42%) control patients (odds ratio 0.43, 0.21 to 0.89, P=0.02). These patterns persisted at six months but were not statistically significant. Patients in the intervention group were more likely to report satisfaction with a range of aspects of services. CONCLUSION: The functional outcome and satisfaction of patients with stroke can be improved by a brief occupational therapy programme carried out in the patient's home immediately after discharge. Major benefits may not, however, be sustained.

Improvement of accuracy in a high-capacity, six degree-of-freedom load cell: application to robotic testing of musculoskeletal joints.

This study investigated a previously unaccounted for source of error in a high-capacity, six degree-of-freedom load cell used in multi-degree-of-freedom robotic testing of musculoskeletal joints, an application requiring a load cell with high accuracy in addition to high load capacity. A method of calibration is presented for reducing the error caused by changes in universal force-moment sensor (UFS) orientation within a gravitational field. Uncorrected, this error can exceed a magnitude of 1% of the full-scale load capacity-the manufacturer-stated accuracy of the UFS. Implementation of the calibration protocol reduced this error by approximately 75% for a variety of loading conditions. This improvement in load cell accuracy (while maintaining full load capacity) should improve both the measurement and control of specimen kinetics by robotic/UFS and other biomechanical testing systems.

Modulation of the biologic activity of the rabbit intervertebral disc by gene therapy: an in vivo study of adenovirus-mediated transfer of the human transforming growth factor beta 1 encoding gene.

STUDY DESIGN: In vivo studies using a rabbit model to determine the biologic effects of direct, adenovirus-mediated transfer of a therapeutic gene to the intervertebral disc. OBJECTIVES: 1) To deliver an exogenous therapeutic gene to rabbit lumbar intervertebral discs in vivo, 2) to quantify the resulting amount of gene expression, and 3) to determine the effect on the biologic activity of the discs. SUMMARY OF BACKGROUND DATA: Although growth factors such as transforming growth factor beta 1 appear to have promising therapeutic properties, there currently is no practical method for sustained delivery of exogenous growth factors to the disc for the management of certain chronic types of disease (e.g., disc degeneration). A possible solution is to modify the disc cells genetically through gene transfer such that the cells manufacture the desired growth factors endogenously on a continuous basis. METHODS: Saline, with or without virus, was injected directly into lumbar discs of 22 skeletally mature female New Zealand white rabbits. Group 1 (n = 11) received the adenovirus construct Ad/CMV-hTGF beta 1 containing the therapeutic human transforming growth factor beta 1-encoding gene. Group 2 (n = 6) received adenovirus containing the luciferase marker gene. Group 3 (n = 5) received saline only. The rabbits were killed 1 week after injection. Immunohistochemical staining for human transforming growth factor beta 1 was performed on the disc tissues of one rabbit from Group 1. Nucleus pulposus tissues from the remaining rabbits were cultured in serumless medium. Bioassays were performed to determine human transforming growth factor beta 1 production and proteoglycan synthesis. RESULTS: Discs injected with Ad/CMV-hTGF beta 1 exhibited extensive and intense positive immunostaining for transforming growth factor beta 1. The nucleus pulposus tissues from the discs injected with Ad/CMV-hTGF beta 1 exhibited a 30-fold increase in active transforming growth factor beta 1 production, and a 5-fold increase in total (active + latent) transforming growth factor beta 1 production over that from intact control discs (P < 0.05). Furthermore, these tissues exhibited a 100% increase in proteoglycan synthesis compared with intact control tissue, which was statistically significant (P < 0.05). CONCLUSIONS: The results of this study suggest that the intervertebral disc is an appropriate site for adenovirus-mediated transfer of exogenous genes and subsequent production of therapeutic growth factors. Gene therapy therefore may have useful applications for study of the basic science of the intervertebral disc and for clinical management of degenerative disc disease.

Adenovirus-mediated gene transfer to nucleus pulposus cells. Implications for the treatment of intervertebral disc degeneration.

STUDY DESIGN: In vitro and in vivo studies using a rabbit model were performed to determine the feasibility of adenovirus-mediated gene transfer to the intervertebral disc. OBJECTIVES: This study was conducted to determine whether it is possible to transfer genes to cells within the intervertebral disc by direct injection of an adenovirus and to determine the duration of gene expression obtained by this method. SUMMARY OF BACKGROUND DATA: Although growth factors have the potential to stimulate the regeneration of nucleus pulposus, sustained delivery of growth factors to a degenerated disc is clinically unfeasible with present technology. Novel approaches such as gene transfer should be investigated as possible solutions to this problem. METHODS: The lacZ marker gene was used to evaluate gene delivery to cells within intervertebral discs. For the in vitro study, cell cultures were established from the nucleus pulposus tissue of New Zealand white rabbits and infected with an adenovirus encoding the lacZ gene (Ad-lacZ). For the in vivo study, the anterior aspects of lumbar intervertebral discs were surgically exposed, and Ad-lacZ in saline solution was directly injected into the nucleus pulposus. An equal volume of saline only was injected into control discs. Expression of the transferred gene was detected by staining with 5-bromo-4-chloro-3-indolyl-beta-galactosidase (X-Gal). RESULTS: The in vitro experiments confirmed that nucleus pulposus cells were efficiently transduced by an adenoviral vector carrying the lacZ gene. In vivo injection of Ad-lacZ into the nucleus pulposus resulted in the transduction of a considerable number of cells. Marker gene expression in vivo persisted at an apparently undiminished level for at least 12 weeks. No staining was noted in control discs. CONCLUSIONS: The results show the feasibility of adenovirus-mediated gene transfer to the intervertebral disc. Expression of the marker gene persisted at least 12 weeks in vivo. This successful demonstration of exogenous gene transfer to the disc and sustained, long-term expression suggests that the adenoviral vector may be suitable for delivery of appropriate genes to the disc for the treatment of spinal disorders.