Tissue-transplant fusion and vascularization of myocardial microtissues and macrotissues implanted into chicken embryos and rats

Cell-based therapies and tissue engineering initiatives are gathering clinical momentum for next-generation treatment of tissue deficiencies. By using gravity-enforced self-assembly of monodispersed primary cells, we have produced adult and neonatal rat cardiomyocyte-based myocardial microtissues that could optionally be vascularized following coating with human umbilical vein endothelial cells (HUVECs). Within myocardial microtissues, individual cardiomyocytes showed native-like cell shape and structure, and established electrochemical coupling via intercalated disks. This resulted in the coordinated beating of microtissues, which was recorded by means of a multi-electrode complementary metal-oxide-semiconductor microchip. Myocardial microtissues (microm3 scale), coated with HUVECs and cast in a custom-shaped agarose mold, assembled to coherent macrotissues (mm3 scale), characterized by an extensive capillary network with typical vessel ultrastructures. Following implantation into chicken embryos, myocardial microtissues recruited the embryo's capillaries to functionally vascularize the rat-derived tissue implant. Similarly, transplantation of rat myocardial microtissues into the pericardium of adult rats resulted in time-dependent integration of myocardial microtissues and co-alignment of implanted and host cardiomyocytes within 7 days. Myocardial microtissues and custom-shaped macrotissues produced by cellular self-assembly exemplify the potential of artificial tissue implants for regenerative medicine.

Glucocorticosteroid-induced spinal osteoporosis: scientific update on pathophysiology and treatment

Glucocorticosteroid-induced spinal osteoporosis (GIOP) is the most frequent of all secondary types of osteoporosis. The understanding of the pathophysiology of glucocorticoid (GC) induced bone loss is of crucial importance for appropriate treatment and prevention of debilitating fractures that occur predominantly in the spine. GIOP results from depressed bone formation due to lower activity and higher death rate of osteoblasts on the one hand, and from increase bone resorption due to prolonged lifespan of osteoclasts on the other. In addition, calcium/phosphate metabolism may be disturbed through GC effects on gut, kidney, parathyroid glands and gonads. Therefore, therapeutic agents aim at restoring balanced bone cell activity by directly decreasing apoptosis rate of osteoblasts (e.g., cyclical parathyroid hormone) or by increasing apoptosis rate of osteoclasts (e.g., bisphosphonates). Other therapeutical efforts aim at maintaining/restoring calcium/phosphate homeostasis: improving intestinal calcium absorption (using calcium supplementation, vitamin D and derivates) and avoiding increased urinary calcium loss (using thiazides) prevent or counteract a secondary hyperparthyroidism. Bisphosphonates, particularly the aminobisphosphonates risedronate and alendronate, have been shown to protect patients on GCs from (further) bone loss to reduce vertebral fracture risk. Calcitonin may be of interest in situation where bisphosphonates are contraindicated or not applicable and in cases where acute pain due to vertebral fracture has to be manage. The intermittent administration of 1-34-parathormone may be an appealing treatment alternative, based on its documented anabolic effects on bone resulting from the reduction of osteoblastic apoptosis. Calcium and vitamin D should be a systematic adjunctive measure to any drug treatment for GIOP. Based on currently available evidence, fluoride, androgens, estrogens (opposed or unopposed) cannot be recommended for the prevention and treatment of GIOP. However, substitution of gonadal hormones may be indicated if GC-induced hypogonadism is present and leads to clinical symptoms. Data using the SERM raloxifene to treat or prevent GIOP are lacking, as are data using the promising bone anabolic agent strontium ranelate. Kyphoplasty performed in appropriately selected osteoporotic patients with painful vertebral fractures is a promising addition to current medical treatment.

The long-term mechanical integrity of non-reinforced PEEK-OPTIMA polymer for demanding spinal applications: experimental and finite-element analysis

Polyetheretherketone (PEEK) is a novel polymer with potential advantages for its use in demanding orthopaedic applications (e.g. intervertebral cages). However, the influence of a physiological environment on the mechanical stability of PEEK has not been reported. Furthermore, the suitability of the polymer for use in highly stressed spinal implants such as intervertebral cages has not been investigated. Therefore, a combined experimental and analytical study was performed to address these open questions. A quasi-static mechanical compression test was performed to compare the initial mechanical properties of PEEK-OPTIMA polymer in a dry, room-temperature and in an aqueous, 37 degrees C environment (n=10 per group). The creep behaviour of cylindrical PEEK polymer specimens (n=6) was measured in a simulated physiological environment at an applied stress level of 10 MPa for a loading duration of 2000 hours (12 weeks). To compare the biomechanical performance of different intervertebral cage types made from PEEK and titanium under complex loading conditions, a three-dimensional finite element model of a functional spinal unit was created. The elastic modulus of PEEK polymer specimens in a physiological environment was 1.8% lower than that of specimens tested at dry, room temperature conditions (P<0.001). The results from the creep test showed an average creep strain of less than 0.1% after 2000 hours of loading. The finite element analysis demonstrated high strain and stress concentrations at the bone/implant interface, emphasizing the importance of cage geometry for load distribution. The stress and strain maxima in the implants were well below the material strength limits of PEEK. In summary, the experimental results verified the mechanical stability of the PEEK-OPTIMA polymer in a simulated physiological environment, and over extended loading periods. Finite element analysis supported the use of PEEK-OPTIMA for load-bearing intervertebral implants.

Short anterior correction of the thoracolumbar/lumbar curve in King 1 idiopathic scoliosis: the behaviour of the instrumented and non-instrumented curves and the trunk balance

This is a retrospective clinical, radiological and patient outcome assessment of 21 consecutive patients with King 1 idiopathic adolescent scoliosis treated by short anterior selective fusion of the major thoracolumbar/lumbar (TL/L) curve. Three-dimensional changes of both curves, changes in trunk balance and rib hump were evaluated. The minimal follow-up was 24 months (max. 83). The Cobb angle of the TL/L curve was 52 degrees (45-67 degrees) with a flexibility of 72% (40-100%). The average length of the main curve was 5 (3-8) segments. An average of 3 (2-4) segments was fused using rigid single rod implants with side-loading screws. The Cobb angle of the thoracic curve was 33 degrees (18-50 degrees) with a flexibility of 69% (29-100%). The thoracic curve in bending was less than 20 degrees in 17 patients, and 20-25 degrees in 4 patients. In the TL/L curve there was an improvement of the Cobb angle of 67%, of the apex vertebral rotation of 51% and of the apex vertebral translation of 74%. The Cobb angle of the thoracic curve improved 29% spontaneously. Shoulder balance improved significantly from an average preoperative imbalance of 14.5-3.1 mm at the last follow-up. Seventy-five percent of the patients with preoperative positive shoulder imbalance (higher on the side of the thoracic curve) had levelled shoulders at the last follow-up. C7 offset improved from a preoperative 19.8 (0-40) to 4.8 (0-18) mm at the last follow-up. There were no significant changes in rotation, translation of the thoracic curve and the clinical rib hump. There were no significant changes in thoracic kyphosis or lumbar lordosis. The average score of the SRS-24 questionnaire at the last follow-up was 91 points (max. 120). We conclude that short anterior selective fusion of the TL/L curve in King 1 scoliosis with a thoracic curve bending to 25 degrees or less (Type 5 according to Lenke classification) results in a satisfactory correction and a balanced spine. Short fusions leave enough mobile lumbar segments for the establishment of global spinal balance. A positive shoulder imbalance is not a contraindication for this procedure. Structural interbody grafts are not necessary to maintain lumbar lordosis.

Harvesting local cylinder autograft from adjacent vertebral body for anterior lumbar interbody fusion: surgical technique, operative feasibility and preliminary clinical results

Autogenous iliac crest has long served as the gold standard for anterior lumbar arthrodesis although added morbidity results from the bone graft harvest. Therefore, femoral ring allograft, or cages, have been used to decrease the morbidity of iliac crest bone harvesting. More recently, an experimental study in the animal showed that harvesting local bone from the anterior vertebral body and replacing the void by a radio-opaque beta-tricalcium phosphate plug was a valid concept. However, such a concept precludes theoretically the use of posterior pedicle screw fixation. At one institution a consecutive series of 21 patients underwent single- or multiple-level circumferential lumbar fusion with anterior cages and posterior pedicle screws. All cages were filled with cancellous bone harvested from the adjacent vertebral body, and the vertebral body defect was filled with a beta-tricalcium phosphate plug. The indications for surgery were failed conservative treatment of a lumbar degenerative disc disease or spondylolisthesis. The purpose of this study, therefore, was to report on the surgical technique, operative feasibility, safety, benefits, and drawbacks of this technique with our primary clinical experience. An independent researcher reviewed all data that had been collected prospectively from the onset of the study. The average age of the patients was 39.9 (26-57) years. Bone grafts were successfully harvested from 28 vertebral bodies in all but one patient whose anterior procedure was aborted due to difficulty in freeing the left common iliac vein. This case was converted to a transforaminal interbody fusion (TLIF). There was no major vascular injury. Blood loss of the anterior procedure averaged 250 ml (50-350 ml). One tricalcium phosphate bone plug was broken during its insertion, and one endplate was broken because of wrong surgical technique, which did not affect the final outcome. One patient had a right lumbar plexopathy that was not related to this special technique. There was no retrograde ejaculation, infection or pseudoarthrosis. One patient experienced a deep venous thrombosis. At the last follow up (mean 28 months) all patients had a solid lumbar spine fusion. At the 6-month follow up, the pain as assessed on the visual analog scale (VAS) decreased from 6.9 to 4.5 (33% decrease), and the Oswestry disability index (ODI) reduced from 48.0 to 31.7 with a 34% reduction. However, at 2 years follow up there was a trend for increase in the ODI (35) and VAS (5). The data in this study suggest that harvesting a cylinder of autograft from the adjacent vertebral body is safe and efficient. Filling of the void defect with a beta-tricalcium phosphate plug does not preclude the use of posterior pedicle screw stabilization.

The treatment of spasticity with Delta9-tetrahydrocannabinol in persons with spinal cord injury

STUDY DESIGN: Open label study to determine drug dose for a randomized double-blind placebo-controlled parallel study. OBJECTIVES: To assess the efficacy and side effects of oral Delta(9)-tetrahydrocannabinol (THC) and rectal THC-hemisuccinate (THC-HS) in SCI patients. SETTING: REHAB Basel, Switzerland. METHOD: Twenty-five patients with SCI were included in this three-phase study with individual dose adjustment, each consisting of 6 weeks. Twenty-two participants received oral THC open label starting with a single dose of 10 mg (Phase 1, completed by 15 patients). Eight subjects received rectal THC-HS (Phase 2, completed by seven patients). In Phase 3, six patients were treated with oral THC and seven with placebo. Major outcome parameters were the spasticity sum score (SSS) using the Modified Ashworth Scale (MAS) and self-ratings of spasticity. RESULTS: Mean daily doses were 31 mg with THC and 43 mg with THC-HS. Mean SSS for THC decreased significantly from 16.72 (+/-7.60) at baseline to 8.92 (+/-7.14) on day 43. Similar improvement was seen with THC-HS. We observed a significant improvement of SSS with active drug (P=0.001) in the seven subjects who received oral THC in Phase 1 and placebo in Phase 3. Major reasons for drop out were increase of pain and psychological side effects. CONCLUSION: THC is an effective and safe drug in the treatment of spasticity. At least 15-20 mg per day were needed to achieve a therapeutic effect.

Synergistic inhibition in cell-cell fusion mediated by the matrix and nucleocapsid protein of canine distemper virus

Canine distemper virus (CDV) causes a chronic, demyelinating, progressive or relapsing neurological disease in dogs, because CDV persists in the CNS. Persistence of virulent CDV, such as the A75/17 strain has been reproduced in cell cultures where it is associated with a non-cytolytic infection with very limited cell-cell fusion. This is in sharp contrast to attenuated CDV infection in cell cultures, such as the Onderstepoort (OP) CDV strain, which produces extensive fusion activity and cytolysis. Fusion efficiency may be determined by the structure of the viral fusion protein per se but also by its interaction with other structural proteins of CDV. This was studied by combining genes derived from persistent and non-persistent CDV strains in transient transfection experiments. It was found that fusion efficiency was markedly attenuated by the structure of the fusion protein of the neurovirulent A75/17-CDV. Moreover, we showed that the interaction of the surface glycoproteins with the M protein of the persistent strain greatly influenced fusion activity. Site directed mutagenesis showed that the c-terminus of the M protein is of particular importance in this respect. Interestingly, although the nucleocapsid protein alone did not affect F/H-induced cell-cell fusion, maximal inhibition occurred when the latter was added to combined glycoproteins with matrix protein. Thus, the present study suggests that very limited fusogenicity in virulent CDV infection, which favours persistence by limiting cell destruction involves complex interactions between all viral structural proteins.

Signal peptide and helical bundle domains of virulent canine distemper virus fusion protein restrict fusogenicity

Persistence in canine distemper virus (CDV) infection is correlated with very limited cell-cell fusion and lack of cytolysis induced by the neurovirulent A75/17-CDV compared to that of the cytolytic Onderstepoort vaccine strain. We have previously shown that this difference was at least in part due to the amino acid sequence of the fusion (F) protein (P. Plattet, J. P. Rivals, B. Zuber, J. M. Brunner, A. Zurbriggen, and R. Wittek, Virology 337:312-326, 2005). Here, we investigated the molecular mechanisms of the neurovirulent CDV F protein underlying limited membrane fusion activity. By exchanging the signal peptide between both F CDV strains or replacing it with an exogenous signal peptide, we demonstrated that this domain controlled intracellular and consequently cell surface protein expression, thus indirectly modulating fusogenicity. In addition, by serially passaging a poorly fusogenic virus and selecting a syncytium-forming variant, we identified the mutation L372W as being responsible for this change of phenotype. Intriguingly, residue L372 potentially is located in the helical bundle domain of the F(1) subunit. We showed that this mutation drastically increased fusion activity of F proteins of both CDV strains in a signal peptide-independent manner. Due to its unique structure even among morbilliviruses, our findings with respect to the signal peptide are likely to be specifically relevant to CDV, whereas the results related to the helical bundle add new insights to our growing understanding of this class of F proteins. We conclude that different mechanisms involving multiple domains of the neurovirulent A75/17-CDV F protein act in concert to limit fusion activity, preventing lysis of infected cells, which ultimately may favor viral persistence.

Riluzole-induced oscillations in spinal networks

We previously showed in dissociated cultures of fetal rat spinal cord that disinhibition-induced bursting is based on intrinsic spiking, network recruitment, and a network refractory period after the bursts. A persistent sodium current (I(NaP)) underlies intrinsic spiking, which, by recurrent excitation, generates the bursting activity. Although full blockade of I(NaP) with riluzole disrupts such bursting, the present study shows that partial blockade of I(NaP) with low doses of riluzole maintains bursting activity with unchanged burst rate and burst duration. More important, low doses of riluzole turned bursts composed of persistent activity into bursts composed of oscillatory activity at around 5 Hz. In a search for the mechanisms underlying the generation of such intraburst oscillations, we found that activity-dependent synaptic depression was not changed with low doses of riluzole. On the other hand, low doses of riluzole strongly increased spike-frequency adaptation and led to early depolarization block when bursts were simulated by injecting long current pulses into single neurons in the absence of fast synaptic transmission. Phenytoin is another I(NaP) blocker. When applied in doses that reduced intrinsic activity by 80-90%, as did low doses of riluzole, it had no effect either on spike-frequency adaptation or on depolarization block. Nor did phenytoin induce intraburst oscillations after disinhibition. A theoretical model incorporating a depolarization block mechanism could reproduce the generation of intraburst oscillations at the network level. From these findings we conclude that riluzole-induced intraburst oscillations are a network-driven phenomenon whose major accommodation mechanism is depolarization block arising from strong sodium channel inactivation.

Lumbar spinal 'juxtaarticular' cyst in a Gordon setter

An 11-year-old Gordon setter bitch was presented with a history of progressive weakness in the right hind limb associated with pain in the lumbar spine. Neurological deficits consisted of ataxia, monoparesis, muscle atrophy and spontaneous over-knuckling of the affected limb. A large 'juxtaarticular' cyst located in a right dorsolateral position of the intervertebral foramen at L3-L4 was diagnosed by magnetic resonance imaging. The cyst was removed through a modified laminectomy. The dog recovered quickly and returned to the owners 4 days after surgery with slight neurological symptoms. During the follow-up examination 2 and 6 months later, the Setter showed normal gait and neurological examination.

Cardiac image fusion from stand-alone SPECT and CT: clinical experience

Myocardial perfusion imaging with SPECT (SPECT-MPI) and 64-slice CT angiography (CTA) are both established techniques for the noninvasive evaluation of coronary artery disease (CAD). Three-dimensional (3D) SPECT/CT image fusion may offer an incremental diagnostic value by integrating both sets of information. We report our first clinical experiences with fused 3D SPECT/CT in CAD patients. METHODS: Thirty-eight consecutive patients with at least 1 perfusion defect on SPECT-MPI (1-d adenosine stress/rest SPECT with (99m)Tc-tetrofosmin) and 64-slice CTA were included. 3D volume-rendered fused SPECT/CT images were generated and compared with the findings from the side-by-side analysis with regard to coronary lesion interpretation by assigning the perfusion defects to their corresponding coronary lesion. RESULTS: The fused SPECT/CT images added information on pathophysiologic lesion severity in 27 coronary stenoses (22%) of 12 patients (29%) (P<0.001). Among 40 equivocal lesions on side-by-side analysis, the fused interpretation confirmed hemodynamic significance in 14 lesions and excluded functional relevance in 10 lesions. In 3 lesions, assignment of perfusion defect and coronary lesion appeared to be reliable on side-by-side analysis but proved to be inaccurate on fused interpretation. Added diagnostic information by SPECT/CT was more commonly found in patients with stenoses of small vessels (P=0.004) and involvement of diagonal branches (P=0.01). CONCLUSION: In addition to being intuitively convincing, 3D SPECT/CT fusion images in CAD may provide added diagnostic information on the functional relevance of coronary artery lesions.

Multiple spinal extradural meningeal cysts presenting as acute paraplegia. Case report and review of the literature

Multiple spinal extradural meningeal cysts are rare. To the authors' knowledge, there have been only four reported cases in the world literature. The authors report a case of multiple spinal extradural meningeal cysts in a 31-year-old woman presenting with acute paraplegia. Magnetic resonance imaging of the thoracolumbar spine revealed multiple extradural cystic lesions extending from T-7 to T-8 and from T-12 to L-3. Intraoperative findings demonstrated a white, fibrous, and tense cyst filled with cerebrospinal fluid-like colorless fluid. Excision of the posterior wall of the symptomatic cyst was followed by immediate neurological improvement. The examination of the pathological specimen showed a thick duralike layer of collagen and an inner membrane of arachnoid that is often not found in these lesions. The final diagnosis was based on combined imaging, intraoperative, and histopathological findings. The authors review the literature and discuss the etiological, diagnostic, and therapeutic aspects of this lesion.

Spinal muscular atrophy: SMN2 pre-mRNA splicing corrected by a U7 snRNA derivative carrying a splicing enhancer sequence

Spinal muscular atrophy (SMA) is a lethal hereditary disease caused by homozygous deletion/inactivation of the survival of motoneuron 1 (SMN1) gene. The nearby SMN2 gene, despite its identical coding capacity, is only an incomplete substitute, because a single nucleotide difference impairs the inclusion of its seventh exon in the messenger RNA (mRNA). This splicing defect can be corrected (transiently) by specially designed oligonucleotides. Here we have developed a more permanent correction strategy based on bifunctional U7 small nuclear RNAs (snRNAs). These carry both an antisense sequence that allows specific binding to exon 7 and a splicing enhancer sequence that will improve the recognition of the targeted exon. When expression cassettes for these RNAs are stably introduced into cells, the U7 snRNAs become incorporated into small nuclear ribonucleoprotein (snRNP) particles that will induce a durable splicing correction. We have optimized this strategy to the point that virtually all SMN2 pre-mRNA becomes correctly spliced. In fibroblasts from an SMA patient, this approach induces a prolonged restoration of SMN protein and ensures its correct localization to discrete nuclear foci (gems).