Early effects of carbachol on the morphology of motor endplates of mammalian skeletal muscle fibers

Long-term disturbance of the calcium homeostasis of motor endplates (MEPs) causes necrosis of muscle fibers. The onset of morphological changes in response to this disturbance, particularly in relation to the fiber type, is presently unknown. Omohyoid muscles of mice were incubated for 1-30 minutes in 0.1 mM carbachol, an acetylcholine agonist that causes an inward calcium current. In these muscles, the structural changes of the sarcomeres and the MEP sarcoplasm were evaluated at the light- and electron-microscopic level. Predominantly in type I fibers, carbachol incubation resulted in strong contractures of the sarcomeres underlying the MEPs. Owing to these contractures, the usual beret-like form of the MEP-associated sarcoplasm was deformed into a mushroom-like body. Consequently, the squeezed MEPs partially overlapped the adjacent muscle fiber segments. There are no signs of contractures below the MEPs if muscles were incubated in carbachol in calcium-free Tyrode's solution. Carbachol induced inward calcium current and produced fiber-type-specific contractures. This finding points to differences in the handling of calcium in MEPs. Possible mechanisms for these fiber-type-specific differences caused by carbachol-induced calcium entry are assessed.

Preparation of intact bovine tail intervertebral discs for organ culture

The intervertebral disc (IVD) is the joint of the spine connecting vertebra to vertebra. It functions to transmit loading of the spine and give flexibility to the spine. It composes of three compartments: the innermost nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous endplates connecting the NP and AF to the vertebral body on both sides. Discogenic pain possibly caused by degenerative intervertebral disc disease (DDD) and disc herniations has been identified as a major problem in our modern society. To study possible mechanisms of IVD degeneration, in vitro organ culture systems with live disc cells are highly appealing. The in vitro culture of intact bovine coccygeal IVDs has advanced to a relevant model system, which allows the study of mechano-biological aspects in a well-controlled physiological and mechanical environment. Bovine tail IVDs can be obtained relatively easy in higher numbers and are very similar to the human lumbar IVDs with respect to cell density, cell population and dimensions. However, previous bovine caudal IVD harvesting techniques retaining cartilaginous endplates and bony endplates failed after 1-2 days of culture since the nutrition pathways were obviously blocked by clotted blood. IVDs are the biggest avascular organs, thus, the nutrients to the cells in the NP are solely dependent on diffusion via the capillary buds from the adjacent vertebral body. Presence of bone debris and clotted blood on the endplate surfaces can hinder nutrient diffusion into the center of the disc and compromise cell viability. Our group established a relatively quick protocol to "crack"-out the IVDs from the tail with a low risk for contamination. We are able to permeabilize the freshly-cut bony endplate surfaces by using a surgical jet lavage system, which removes the blood clots and cutting debris and very efficiently reopens the nutrition diffusion pathway to the center of the IVD. The presence of growth plates on both sides of the vertebral bone has to be avoided and to be removed prior to culture. In this video, we outline the crucial steps during preparation and demonstrate the key to a successful organ culture maintaining high cell viability for 14 days under free swelling culture. The culture time could be extended when appropriate mechanical environment can be maintained by using mechanical loading bioreactor. The technique demonstrated here can be extended to other animal species such as porcine, ovine and leporine caudal and lumbar IVD isolation.

Osseointegration of hollow cylinder based spinal implants in normal and osteoporotic vertebrae: a sheep study

INTRODUCTION: Osteoporosis is not only responsible for an increased number of metaphyseal and spinal fractures but it also complicates their treatment. To prevent the initial loosening, we developed a new implant with an enlarged implant/bone interface based on the concept of perforated, hollow cylinders. We evaluated whether osseointegration of a hollow cylinder based implant takes place in normal or osteoporotic bone of sheep under functional loading conditions during anterior stabilization of the lumbar spine. MATERIALS AND METHODS: Osseointegration of the cylinders and status of the fused segments (ventral corpectomy, replacement with iliac strut, and fixation with testing implant) were investigated in six osteoporotic (age 6.9 +/- 0.8 years, mean body weight 61.1 +/- 5.2 kg) and seven control sheep (age 6.1 +/- 0.2 years, mean body weight 64.9 +/- 5.7 kg). Osteoporosis was introduced using a combination protocol of ovariectomy, high-dose prednisone, calcium and phosphor reduced diet and movement restriction. Osseointegration was quantified using fluorescence and conventional histology; fusion status was determined using biomechanical testing of the stabilized segment in a six-degree-of-freedom loading device as well as with radiological and histological staging. RESULTS: Intact bone trabeculae were found in 70% of all perforations without differences between the two groups (P = 0.26). Inside the cylinders, bone volume/total volume was significantly higher than in the control vertebra (50 +/- 16 vs. 28 +/- 13%) of the same animal (P<0.01), but significantly less (P<0.01) than in the near surrounding (60 +/- 21%). After biomechanical testing as described in Sect. "Materials and methods", seven spines (three healthy and four osteoporotic) were classified as completely fused and six (four healthy and two osteoporotic) as not fused after a 4-month observation time. All endplates were bridged with intact trabeculae in the histological slices. CONCLUSIONS: The high number of perforations, filled with intact trabeculae, indicates an adequate fixation; bridging trabeculae between adjacent endplates and tricortical iliac struts in all vertebrae indicates that the anchorage is adequate to promote fusion in this animal model, even in the osteoporotic sheep.

Cement distribution, volume, and compliance in vertebroplasty: some answers from an anatomy-based nonlinear finite element study

STUDY DESIGN: The biomechanics of vertebral bodies augmented with real distributions of cement were investigated using nonlinear finite element (FE) analysis. OBJECTIVES: To compare stiffness, strength, and stress transfer of augmented versus nonaugmented osteoporotic vertebral bodies under compressive loading. Specifically, to examine how cement distribution, volume, and compliance affect these biomechanical variables. SUMMARY OF BACKGROUND DATA: Previous FE studies suggested that vertebroplasty might alter vertebral stress transfer, leading to adjacent vertebral failure. However, no FE study so far accounted for real cement distributions and bone damage accumulation. METHODS: Twelve vertebral bodies scanned with high-resolution pQCT and tested in compression were augmented with various volumes of cements and scanned again. Nonaugmented and augmented pQCT datasets were converted to FE models, with bone properties modeled with an elastic, plastic and damage constitutive law that was previously calibrated for the nonaugmented models. The cement-bone composite was modeled with a rule of mixture. The nonaugmented and augmented FE models were subjected to compression and their stiffness, strength, and stress map calculated for different cement compliances. RESULTS: Cement distribution dominated the stiffening and strengthening effects of augmentation. Models with cement connecting either the superior or inferior endplate (S/I fillings) were only up to 2 times stiffer than the nonaugmented models with minimal strengthening, whereas those with cement connecting both endplates (S + I fillings) were 1 to 8 times stiffer and 1 to 12 times stronger. Stress increases above and below the cement, which was higher for the S + I cases and was significantly reduced by increasing cement compliance. CONCLUSION: The developed FE approach, which accounts for real cement distributions and bone damage accumulation, provides a refined insight into the mechanics of augmented vertebral bodies. In particular, augmentation with compliant cement bridging both endplates would reduce stress transfer while providing sufficient strengthening.

Cellular and network mechanisms of rhythm generation in spinal cord circuits

The generation of rhythmic electrical activity is a prominent feature of spinal cord circuits that is used for locomotion and also for circuit refinement during development. The mechanisms involved in rhythm generation in spinal cord networks are not fully understood. It is for example not known whether spinal cord rhythms are driven by pacemaker neurons and if yes, which neurons are involved in this function. We studied the mechanisms involved in rhythm generation in slice cultures from fetal rats that were grown on multielectrode arrays (MEAs). We combined multisite extracellular recordings from the MEA electrodes with intracellular patch clamp recordings from single neurons. We found that spatially restricted oscillations of activity appeared in most of the cultures spontaneously. Such activity was based on intrinsic activity in a percentage of the neurons that could activate the spinal networks through recurrent excitation. The local oscillator networks critically involved NMDA, AMPA and GABA / glycine receptors at subsequent phases of the oscillation cycle. Intrinsic spiking in individual neurons (in the absence of functional synaptic coupling) was based on persistent sodium currents. Intrinsic firing as well as persistent sodium currents were increased by 5-HT through 5-HT2 receptors. Comparing neuronal activity to muscle activity in co-cultures of spinal cord slices with muscle fibers we found that a percentage of the intrinsically spiking neurons were motoneurons. These motoneurons were electrically coupled among each other and they could drive the spinal networks through cholinergic recurrent excitation. These findings open the possibility that during development rhythmic activity in motoneurons is not only involved in circuit refinement downstream at the neuromuscular endplates but also upstream at the level of spinal cord circuits.

Imaging of chronic recurrent multifocal osteomyelitis of childhood first presenting with isolated primary spinal involvement

OBJECTIVE: Initial presentation with primary spinal involvement in chronic recurrent multifocal osteomyelitis of childhood (CRMO) is rare. Our objective was to review the imaging appearances of three patients who had CRMO who initially presented with isolated primary spinal involvement. DESIGN AND PATIENTS: The imaging, clinical, laboratory and histology findings of the three patients were retrospectively reviewed. Imaging included seven spinal MR imaging scans, one computed tomography scan, nine bone scans, two tomograms and 16 radiographs. These were reviewed by two musculoskeletal radiologists and a consensus view is reported. All three patients presented with atraumatic spinal pain and had extensive bone spinal pathology. The patients were aged 11, 13 and 12 years. There were two females and one male. RESULTS AND CONCLUSIONS: The initial patient had thoracic T6 and T8 vertebra plana. Bone scan showed additional vertebral body involvement. Follow-up was available over a 3 year period. The second patient had partial collapse of T9 and, 2 years later, of C6. Subsequently extensive multifocal disease ensued and follow-up was available over 8 years. The third patient initially had L3 inferior partial collapse and 1 year later T8 involvement with multifocal disease. Follow-up was available over 3 years. The imaging findings of the three patients include partial and complete vertebra plana with a subchondral line adjacent to endplates associated with bone marrow MR signal alterations. Awareness of the imaging appearances may help the radiologist to include this entity in the differential diagnosis in children who present with spinal pathology and no history of trauma. Histopathological examination excludes tumor and infection but with typical imaging findings may not always be necessary.

Embedding of human vertebral bodies leads to higher ultimate load and altered damage localisation under axial compression

Computer tomography (CT)-based finite element (FE) models of vertebral bodies assess fracture load in vitro better than dual energy X-ray absorptiometry, but boundary conditions affect stress distribution under the endplates that may influence ultimate load and damage localisation under post-yield strains. Therefore, HRpQCT-based homogenised FE models of 12 vertebral bodies were subjected to axial compression with two distinct boundary conditions: embedding in polymethylmethalcrylate (PMMA) and bonding to a healthy intervertebral disc (IVD) with distinct hyperelastic properties for nucleus and annulus. Bone volume fraction and fabric assessed from HRpQCT data were used to determine the elastic, plastic and damage behaviour of bone. Ultimate forces obtained with PMMA were 22% higher than with IVD but correlated highly (R2 = 0.99). At ultimate force, distinct fractions of damage were computed in the endplates (PMMA: 6%, IVD: 70%), cortex and trabecular sub-regions, which confirms previous observations that in contrast to PMMA embedding, failure initiated underneath the nuclei in healthy IVDs. In conclusion, axial loading of vertebral bodies via PMMA embedding versus healthy IVD overestimates ultimate load and leads to distinct damage localisation and failure pattern.

Assessment of the Matrix Degenerative Effects of MMP-3, ADAMTS-4 and HTRA1 injected into a bovine Intervertebral Disc Organ Culture Model

Study Design. In vitro study to develop an intervertebral disc degeneration (IDD) organ culture model, using coccygeal bovine intervertebral discs (IVDs) and injection of proteolytic enzymes MMP-3, ADAMTS-4 and HTRA1.Objective. This study aimed to develop an in-vitro model of enzyme-mediated IDD to mimic the clinical outcome in humans for investigation of therapeutic treatment options.Summary of Background Data. Bovine IVDs are comparable to human IVDs in terms of cell composition and biomechanical behavior. Researchers injected papain and trypsin into them to create an IDD model with a degenerated nucleus pulposus (NP) area. They achieved macroscopic cavities as well as a loss of glycosaminoglycans (GAGs). However, none of these enzymes are clinically relevant.Methods. Bovine IVDs were harvested maintaining the endplates. Active forms of MMP-3, ADAMTS-4 and HTRA1 were injected at a dose of 10μg/ml each. Phosphate buffered saline (PBS) was injected as a control. Discs were cultured for 8 days and loaded diurnally (day 1 to day 4 with 0.4 MPa for 16 h) and left under free swelling condition from day 4 to day 8 to avoid expected artifacts due to dehydration of the NP. Outcome parameters included disc height, metabolic cell activity, DNA content, glycosaminoglycan (GAG) content, total collagen content, relative gene expression and histological investigation.Results. The mean metabolic cell activity was significantly lower in the NP area of discs injected with ADAMTS-4 compared to the day 0 control discs. Disc height was decreased following injection with HTRA1, and was significantly correlated with changes in GAG/DNA of the NP tissue. Total collagen content tended to be lower in groups injected with ADAMTS4 and MMP-3.Conclusion. MMP-3, ADAMTS-4 and HTRA1 neither provoked visible matrix degradation nor major shifts in gene expression. However, cell activity was significantly reduced and HTRA1 induced loss of disc height which positively correlated with changes in GAG/DNA content. The use of higher doses of these enzymes or a combination thereof may therefore be necessary to induce disc degeneration

An experimentally validated finite element method for augmented vertebral bodies

Background Finite element models of augmented vertebral bodies require a realistic modelling of the cement infiltrated region. Most methods published so far used idealized cement shapes or oversimplified material models for the augmented region. In this study, an improved, anatomy-specific, homogenized finite element method was developed and validated to predict the apparent as well as the local mechanical behavior of augmented vertebral bodies. Methods Forty-nine human vertebral body sections were prepared by removing the cortical endplates and scanned with high-resolution peripheral quantitative CT before and after injection of a standard and a low-modulus bone cement. Forty-one specimens were tested in compression to measure stiffness, strength and contact pressure distributions between specimens and loading-plates. From the remaining eight, fourteen cylindrical specimens were extracted from the augmented region and tested in compression to obtain material properties. Anatomy-specific finite element models were generated from the CT data. The models featured element-specific, density-fabric-based material properties, damage accumulation, real cement distributions and experimentally determined material properties for the augmented region. Apparent stiffness and strength as well as contact pressure distributions at the loading plates were compared between simulations and experiments. Findings The finite element models were able to predict apparent stiffness (R2 > 0.86) and apparent strength (R2 > 0.92) very well. Also, the numerically obtained pressure distributions were in reasonable quantitative (R2 > 0.48) and qualitative agreement with the experiments. Interpretation The proposed finite element models have proven to be an accurate tool for studying the apparent as well as the local mechanical behavior of augmented vertebral bodies.

Tissue properties of the human vertebral body sub-structures evaluated by means of microindentation

Purpose The better understanding of vertebral mechanical properties can help to improve the diagnosis of vertebral fractures. As the bone mechanical competence depends not only from bone mineral density (BMD) but also from bone quality, the goal of the present study was to investigate the anisotropic indentation moduli of the different sub-structures of the healthy human vertebral body and spondylophytes by means of microindentation. Methods Six human vertebral bodies and five osteophytes (spondylophytes) were collected and prepared for microindentation test. In particular, indentations were performed on bone structural units of the cortical shell (along axial, circumferential and radial directions), of the endplates (along the anterio-posterior and lateral directions), of the trabecular bone (along the axial and transverse directions) and of the spondylophytes (along the axial direction). A total of 3164 indentations down to a maximum depth of 2.5 µm were performed and the indentation modulus was computed for each measurement. Results The cortical shell showed an orthotropic behavior (indentation modulus, Ei, higher if measured along the axial direction, 14.6±2.8 GPa, compared to the circumferential one, 12.3±3.5 GPa, and radial one, 8.3±3.1 GPa). Moreover, the cortical endplates (similar Ei along the antero-posterior, 13.0±2.9 GPa, and along the lateral, 12.0±3.0 GPa, directions) and the trabecular bone (Ei= 13.7±3.4 GPa along the axial direction versus Ei=10.9±3.7 GPa along the transverse one) showed transversal isotropy behavior. Furthermore, the spondylophytes showed the lower mechanical properties measured along the axial direction (Ei=10.5±3.3 GPa). Conclusions The original results presented in this study improve our understanding of vertebral biomechanics and can be helpful to define the material properties of the vertebral substructures in computational models such as FE analysis.