Effect of multiple freeze-thaw cycles on intervertebral dynamic motion characteristics in the porcine lumbar spine

Fresh frozen spine specimens are commonly used in biomechanical investigations of the spine. Since many study designs require staged preparation and testing, the effect of multiple freeze-thaw cycles on motion behavior should be understood. The objective of this study was to investigate the effect of multiple freeze-thaw cycles on the biomechanical parameters measured during dynamic pure moment loading. Ten porcine lumbar motion segments were harvested immediately after death and potted in acrylic fixtures. Specimens were tested in continuous pure moment flexion-extension, lateral flexion, and rotation cycles up to a limit of +/-5Nm. Moment-angular displacement data were analyzed and parameters quantified including range of motion, elastic zone, transitional zone (neutral region) size and slope, and width of the hysteresis loop. All specimens were tested at baseline and after each of three subsequent cycles of freezing and thawing. The transitional zone size decreased and the transitional zone slope increased during flexion-extension and lateral bending after the initial freeze-thaw cycle. These parameters were not altered after subsequent cycles. No significant change was observed in the elastic zone or width of hysteresis loop. Although freezing porcine spine specimens increased the stiffness in the neutral region of motion, up to three subsequent cycles of freezing and thawing did not further affect these motion characteristics. This suggests that data obtained from porcine spines which have been frozen and thawed multiple times are stable after initial freezing.     

Activity of dehydroabietic acid derivatives against wood contaminant fungi

The antifungal activity of 10 dehydroabietic acid derivatives with different configuration in A and B rings (cis/trans A/B junction) and different substituents and/or functionalities was evaluated in bioassays in vitro and in situ (pine wood blocks).

The test compounds dissolved in acetone were assayed at several concentrations w/w (test compound/culture medium) against the fungi. The Relative Inhibition (RI) was determined by measuring the radial growth of colonies of the fungi treated with the test compounds by comparison with those of control cultures; the results are expressed as EC₅₀.

The results of bioassays in vitro have shown that hydroxyl and aldehyde functions are required for antifungal activity in this group of compounds and deisopropylation can increase the activity. Our assay of antifungal activity in situ (in pine wood blocks) provides a means to investigate the preservative activities of these antifungal compounds under actual conditions of use.

The dehydroabietic acid derivative cis-deisopropyldehydroabietanol (10) inhibited the growth of several of the fungi tested, in vitro and in situ.

The results obtained in situ with the test compound (10) at 6% and 8% were not significantly different from the reference products and a good level of protection of the wood against the organisms tested was achieved.

The results in wood bioassays present new possibilities in the search for natural new compounds in the wood protection, as an alternative to conventional fungicides.     

Evaluation of a robot-assisted testing system for multisegmental spine specimens

Mono- and multi-segmental testing methods are required to identify segmental motion patterns and evaluate the biomechanical behaviour of the spine. This study aimed to evaluate a new testing system for multisegmental specimens using a robot combined with an optical motion analysis system. After validation of the robotic system for accuracy, two groups of calf specimens (six monosegmental vs. six multisegmental) were mounted and the functional unit L3-4 was observed. Using rigid body markers, range of motion (ROM), elastic zone (EZ) and neutral zone (NZ), as well as stiffness properties of each functional spine unit (FSU) was acquired by an optical motion capture system. Finite helical axes (FHA) were calculated to analyse segmental movements. Both groups were tested in flexion and extension. A pure torque of 7.5 Nm was applied. Statistical analyses were performed using the Mann-Whitney U-test. Repeatability of robot positioning was -0.001±0.018 mm and -0.025±0.023° for translations and rotations, respectively. The accuracy of the optical system for the proposed set-up was 0.001±0.034 mm for translations and 0.075±0.12° for rotations. No significant differences in mean values and standard deviations of ROM for L3-4 compared to literature data were found. A robot-based facility for testing multisegmental spine units combined with a motion analysis system was proposed and the reliability and reproducibility of all system components were evaluated and validated. The proposed set-up delivered ROM results for mono- and multi-segmental testing that agreed with those reported in the literature. Representing the FHA via piercing points determined from ROM was the first attempt showing a relationship between ROM and FHA, which could facilitate the interpretation of spine motion patterns in the future.     

Estrogen-like effect of a Cimicifuga racemosa extract sub-fraction as assessed by in vivo, ex vivo and in vitro assays

Black cohosh (Cimicifuga racemosa) is used in the treatment of painful menstruation and menopausal symptoms. Data about the nature of the active compounds and mechanism(s) of action are still controversial, chiefly with respect to its estrogenic activity.

This work aimed to assess the possible estrogenic activity of a commercial dry hydro-alcoholic extract of C. racemosa and its hydrophilic and lipophilic sub-fractions on in vivo, ex vivo, and in vitro assays.

In a yeast estrogen screen, only the lipophilic sub-fraction was able to activate the human estrogen receptor , with a lower potency but comparable efficacy to that of 17 β-estradiol.

Neither the total extract nor the lipophilic sub-fraction showed an in vivo uterotrophic effect in 21-day-old rats. Uterine tissues obtained ex vivo from C. racemosa treated animals were generally much less sensitive to oxytocin, prostaglandin F_2, and bradykinin than tissues obtained from estradiol valerate treated rats.

The lipophilic sub-fraction, instead, induced a dose-dependent inhibitory activity on the in vitro response to oxytocin, prostaglandin F_2, and bradykinin of uterine horns from naïve 28-day-old rats, with a potency rate close to 1:30 of that of 17 β-estradiol.

Reported results confirm the effectiveness of C. racemosa in menstrual distress and further emphasize the possibility that lipophilic constituents bind to an as yet not identified estrogen receptor, likely inversely involved in inflammation.     

Identification of [I]endothelin binding sites in human coronary tissue

In vitro receptor autoradiography has been used to study the distribution of [¹²⁵I]endothelin binding sites in human coronary tissue from patients undergoing cardiac transplantation. Dense binding of [¹²⁵I]endothelin was associated with the smooth muscle of epicardial coronary arteries as well as to perivascular regions of these vessels. Binding was also associated with the ventricular myocardium. There was an increased binding of [¹²⁵I]endothelin to atheromatous tissue, both coronary arteries and vein graft.

The [¹²⁵I]endothelin binding sites identified using in vitro autoradiography are likely to be functionally relevant since endothelin causes a concentration-dependent contraction of segments of human epicardial coronary arteries in vitro and also has positive inotropic activity on isolated human cardiomyocytes.

The presence of specific binding sites for [¹²⁵I]endothelin on coronary tissue and the increased binding in atheromatous tissue suggest that endothelin is a peptide which may play a role in the maintenance of vascular tone and/or the pathogenesis of ischaemic heart disease.     

Biomechanicsl evaluation of a stand-alone interbody fusion cage based on porous TiO2/glass-ceramic on the human cervical spine]

Recently, there has been a rapid increase in the use of cervical spine interbody fusion cages, differing in design and biomaterial used, in competition to autologous iliac bone graft and bone cement (PMMA). Limited biomechanical differences in primary stability, as well as advantages and disadvantages of each cage or material have been investigated in studies, using an in vitro human cervical spine model. 20 human cervical spine specimens were tested after fusion with either a cubical stand-alone interbody fusion cage manufactured from a new porous TiO2/glass composite (Ecopore) or PMMA after discectomy. Non-destructive biomechanical testing was performed, including flexion/extension and lateral bending using a spine testing apparatus. Three-dimensional segmental range of motion (ROM) was evaluated using an ultrasound measurement system. ROM increased more in flexion/extension and lateral bending after PMMA fusion (26.5%/36.1%), then after implantation of the Ecopore-cage (8.1%/7.8%). In this first biomechanical in vitro examination of a new porous ceramic bone replacement material a) the feasibility and reproducibility of biomechanical cadaveric cervical examination and its applicability was demonstrated, b) the stability of the ceramic cage as a stand alone interbody cage was confirmed in vitro, and c) basic information and knowledge for our intended biomechanical and histological in vivo testing, after implantation of Ecopore in cervical sheep spines, were obtained.     

Adaptation of a clinical fixation device for biomechanical testing of the lumbar spine

In-vitro biomechanical testing is widely performed for characterizing the load-displacement characteristics of intact, injured, degenerated, and surgically repaired osteoligamentous spine specimens. Traditional specimen fixture devices offer an unspecified rigidity of fixation, while varying in the associated amounts and reversibility of damage to and “coverage” of a specimen – factors that can limit surgical access to structures of interest during testing as well as preclude the possibility of testing certain segments of a specimen. Therefore, the objective of this study was to develop a specimen fixture system for spine biomechanical testing that uses components of clinically available spinal fixation hardware and determine whether the new system provides sufficient rigidity for spine biomechanical testing. Custom testing blocks were mounted into a robotic testing system and the angular deflection of the upper fixture was measured indirectly using linear variable differential transformers. The fixture system had an overall stiffness 37.0, 16.7 and 13.3 times greater than a typical human functional spine unit for the flexion/extension, axial rotation and lateral bending directions respectively – sufficient rigidity for biomechanical testing. Fixture motion when mounted to a lumbar spine specimen revealed average motion of 0.6, 0.6, and 1.5° in each direction. This specimen fixture method causes only minimal damage to a specimen, permits testing of all levels of a specimen, and provides for surgical access during testing.     

Study in situ et in vivo of the acceleration of lumbar vertebrae of a primate exposed to vibration in the Z-axis

In order to study the propagation of vibration along the spine, the authors developed a method allowing in vivo et in situ measurement of vibrational accelerations in the lower lumbar spine of a primate.

The surgical technique used to anchor the miniature accelerometers on the anterior wall of vertebral bodies is described. After recovery, the sitting animal is exposed to a vibration stimulus. Direct recording of data obtained from the lowest four lumbar vertebrae of a healthy live animal provide objective information on the behavior of intervertebral discs and will later permit definition of the role of skeletal muscles in the spinal response to a vibrational stress.     

Detection of Proliferating Cell Nuclear Antigen in Tissues of Three Small Fish Species

An immunohistochemical assay for proliferating cell nuclear antigen (PCNA) identifies cells in all active phases of the cell cycle. In this study, PCNA methodology, which was developed primarily for mammalian tissues, was adapted to three small fish species, medaka (Ory-zias latipes), guppy (Poecilia reticulata), and western mosquitofish (Gambusia affiinis) that are used in carcinogenesis bioassays and environmental sentinel studies. Our study showed that PCNA can be identified in routinely processed, paraffin embedded specimens of these fishes. Optimum staining conditions were dependent on fixative, primary antibody, antigen retrieval processing. and protein blocking reagent. Best results were achieved using 10% neutral buffered formalin as the fixative, clone PC10 as the primary antibody, and a combination of powdered milk and bovine serum albumin as a protein block. Except for medaka specimens, antigen retrieval was not required for specimens preserved in 10% neutral buffered formalin. but was required for the other fixatives tested. In whole fish specimens, PCNA marked cells in normally proliferating tissues such as testis. ovary, primary filament epithelium of the gill, hematopoietic tissues, thymus, retina and alimentary tract. The study demonstrated the successful application of mammalian-based PCNA technology to these aquatic species. Further applications of the assay will aid in understanding the role of cell proliferation in normal, diseased, and toxicant-affected tissues of aquatic animals.     

Neutral zone and range of motion in the spine are greater with stepwise loading than with a continuous loading protocol. An in vitro porcine investigation

Biomechanical testing of the spine has traditionally been performed to help understand the normal function of the spine as well as to evaluate the effects of injury and surgical procedures on spinal behaviour. The overall objective of this investigation was to compare traditional stepwise loading with the recently introduced continuous loading protocol, determining the effect of loading protocol on the mechanical behaviour of the spine. For all tests, a custom spine testing machine was used to apply pure moments of flexion extension, axial rotation, and lateral bending to a maximum of 2 Nm, using six porcine cervical spine specimens (C2-C4). Motions of C2 with respect to C4 were measured with an optoelectronic camera system. Motion parameters calculated were range of motion (ROM), neutral zone (NZ), and the ratio of NZ and ROM. The continuous loading protocol had smaller values for all motion parameters in each loading direction (p<0.05). ROM for the continuous test ranged between 88% and 93% of that of stepwise for the three loading directions. The continuous protocol NZ was 56-75% of that of the stepwise test. The findings of the study demonstrate that the two loading protocols provide differing spinal behaviours.     

Clinical spinal instability and low back pain.

Clinical instability is an important cause of low back pain. Although there is some controversy concerning its definition, it is most widely believed that the loss of normal pattern of spinal motion causes pain and/or neurologic dysfunction. The stabilizing system of the spine may be divided into three subsystems: (1) the spinal column; (2) the spinal muscles; and (3) the neural control unit. A large number of biomechanical studies of the spinal column have provided insight into the role of the various components of the spinal column in providing spinal stability. The neutral zone was found to be a more sensitive parameter than the range of motion in documenting the effects of mechanical destabilization of the spine caused by injury and restabilization of the spine by osteophyle formation, fusion or muscle stabilization. Clinical studies indicate that the application of an external fixator to the painful segment of the spine can significantly reduce the pain. Results of an in vitro simulation of the study found that it was most probably the decrease in the neutral zone, which was responsible for pain reduction. A hypothesis relating the neutral zone to pain has been presented. The spinal muscles provide significant stability to the spine as shown by both in vitro experiments and mathematical models. Concerning the role of neuromuscular control system, increased body sway has been found in patients with low back pain, indicating a less efficient muscle control system with decreased ability to provide the needed spinal stability.     

Fluorescence polarization and pigment orientation in photosynthetic bacteria

Fluorescence polarization of photosynthetic bacteria with various types of chromatophores suggests an orientation of bacteriochlorophylls, but not of carotenoids. Comparison of the in vitro fluorescence polarization spectra of bacteriochlorophyll at high and low concentration and at 77 °K with those in vivo at various temperatures and in the presence of carbowax indicates that dichroism of shape effects the in vivo spectra. Both orientation and shape effects are highest for bacteria containing lamellar-type chromatophores, and lowest in those containing vesicle-type ones.

The polarization values of the bacteria studied are similar for the various red bands, indicating a nearly parallel orientation of the adjacent bacteriochlorophylls.     

The onset and progression of spinal injury: a demonstration of neutral zone sensitivity.

Spinal injuries are a great cost to society and the afflicted individuals. It is well known that most spinal injuries are not bony fractures but rather soft tissue lesions falling in the 'subfailure' region. For the clinical diagnosis of spinal injuries, abnormal motion patterns under physiological loads are considered an important factor. The purpose of the present study was to determine the onset and progression of spinal injury, and compare the sensitivity of three motion parameters: neutral zone (NZ), elastic zone (EZ), and range of motion (ROM). Spinal injury was defined as a significant increase in any of the three motion parameters. A repeatable high-speed flexion-compression load vector was applied individually to six porcine cervical spine specimens. Several impacts of increasing severity were applied to each specimen. After each impact, flexion-extension motion was measured. Neutral zone was the residual deformation from the neutral position to the position under zero load at the start of the final load cycle. Elastic zone was the displacement from zero load to the maximum load on the final load cycle. Range of motion was the sum of the neutral and elastic zones. The first significant increase in motion was determined by the neutral zone parameter with few observable anatomic lesions on the specimens. This was the onset of spinal injury. The next significant motion increase was also determined by the neutral zone parameter. After this motion increase, termed the progression of injury, ligament ruptures were observed in some specimens. It was concluded that the neutral zone was the most sensitive motion parameter in defining the onset and progression of spinal injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

A preliminary characterization of some pectins from quince fruit (Cydonia oblonga Mill.) and prickly pear (Opuntia ficus indica) peel

The preliminary characterization of a hot acid extracted pectin from quince (Cydonia oblonga Mill.) and from prickly pear (Opuntia ficus indica) peel was carried out. The yield of the extraction, the galacturonic acid content and the neutral sugar composition were determined and compared with published data on apple and lemon pectins

The pectin yield from quince was on average 0·53% on fresh weight, which is of a similar order to apple. The quince pectin had a high galacturonic content (about 78%), and a degree of methoxylation of about 59% corresponding to a medium-high methoxyl pectin.

The prickly pear pectin yield was 0·12% on fresh weight. This pectin had a galacturonic acid content of 64%, a low degree of methoxylation (10%), a high acetyl (10%) and neutral sugar content (51% galacturonic). It might be related to similar polygalacturonides present in the mucilages of other Cactaceae.     

Spinal biomechanical properties are significantly altered with a novel embalming method

In vitro tests on the biomechanical properties of human spines are often performed using fresh frozen specimens. However, this carries the risk of pathogen transfer from specimen to the worker and the specimens can only be used for a limited amount of time. Human spinal specimens embalmed with formaldehyde carry an almost absent risk of transfer of pathogens and can be stored and used for a long time, but the tissue properties are strongly affected making this method inapplicable for biomechanical testing. In this study, a new embalming technique called Fix for Life (F4L), which claims to preserve the tissue properties, was tested. The range of motion (ROM) and stiffness of six fresh human spinal specimens was measured using a spinal motion simulator before and after F4L embalming. After F4L embalming, spinal stiffness increased in flexion-extension by 230%, in lateral bending by 284% and in axial rotation by 271%. ROM decreased by 46% in flexion-extension, 56% in lateral bending and 54% in axial rotation. In conclusion, based on this study, F4L does not maintain physiological spinal biomechanical properties, and we propose that this method should not be used for biomechanical studies. Nevertheless, the method may be an alternative to formaldehyde fixation in situations such as training and education because the effect on spinal biomechanics is less detrimental than formaldehyde and tissue color is maintained.     

Resorption und Stoffwechsel von Glucose im Darmtrakt der Wanderheuschrecke Locusta migratoria

Absorption and metabolic fate of ¹⁴C-glucose after injection into mid-gut preparations from Locusta migratoria were studied in vitro. After 5 min nearly half of the total radioactivity had already passed into the incubation medium and in 2 hr there was an increase to 75 per cent. The radioactivity in the medium was found mainly in glucose and fructose, which were approximately equally labelled, and a minor part appeared in the organic acid and sugar phosphate fraction. This pattern of labelling changed drastically when non-radio active glucose was present in the incubation mixture. In these experiments an extensive synthesis of sucrose could be demonstrated.

Sucrose formation from UDP-¹⁴C-glucose by mid-gut homogenates was stimulated by fructose-6-phosphate.

The appearance of radioactivity in the contents of the gut and in the faeces was demonstrated in in vivo experiments after injection of ¹⁴C-glucose into the haemolymph.

Sugar concentrations in the haemolymph of locusts were measured after 24 hr starvation and after feeding. The levels of glucose, fructose, and sucrose were very low in the starving animals and increased ten- to twenty-fold after feeding. The trehalose concentrations remained high and relatively constant.

The activities of 18 enzymes of the intermediary metabolism were assayed in the fore-, mid-, and hindgut.     

Specificity of the action of parathyroid hormone upon mitochondrial metabolism: Cyanogen bromide-derived parathyroid-hormone peptides

The mitochondrial response to cyanogen bromide-treated parathyroid hormone was studied as a means of testing further the relationship between the structure and the effects in vitro of this hormone. The treated hormone and appropriate control hormone were tested in a standard bioassay and in a mitochondrial assay system in vitro.

Reaction of more than 90 % of the methionine residues in the hormone resulted in total inactivation of the hormone both in vivo and in vitro. This result disagrees with previously published data.     

A continuous pure moment loading apparatus for biomechanical testing of multi-segment spine specimens

An apparatus is described that enables the application of continuous pure moment loads to multi-segment spine specimens. This loading apparatus allows continuous cycling of the spine between specified flexion and extension (or right and left lateral bending) maximum load endpoints. Using a six-degree-of-freedom load cell and three-dimensional optoelectronic stereophotogrammetry, characteristic displacement versus load hysteresis curves can be generated and analyzed for different spinal constructs of interest. Unlike quasi-static loading, the use of continuous loading permits the analysis of the spine's behaviour within the neutral zone. This information is of particular clinical significance given that the instability of a spinal segment is related to its flexibility within the neutral zone. Representative curves for the porcine lumbar spine in flexion-extension and lateral bending are presented to illustrate the capabilities of this system.     

Expression of the A12 form of acetylcholinesterase by developing avian leg muscle cells in vivo and during differentiation in primary cell cultures

The accumulation of the molecular forms of acetylcholinesterase (AChE) has been studied in leg muscles during embryonic chick development and in cell cultures initiated with myoblasts obtained from embryos at different stages of development. The collagen-tailed, A₁₂ form appears in leg muscles as soon as day 5 in ovo. An early excision of the lumbar zone of the neural tube at day 2 1/2 in ovo severely delayed the morphological development. In leg muscles dissected at day 12 in ovo from operated embryos, we found that the total amount of AChE activity and particularly the proportion of A₁₂ form were dramatically reduced.

Muscle cells were grown in vitro in a medium supplemented with fetal calf serum. In these conditions, chick muscle cells unequivocally synthesize the A₁₂ form when they originated from muscles which accumulated this form in vivo. In contrast, myoblasts obtained from 5-day old embryo leg muscles did not produce the A₁₂ form either in aneural cultures or in the presence of nerve cells. In relation with previous observations concerning chick myogenesis, we discuss the possibility that this difference reflects the existence of two types of myoblasts. This hypothesis would also explain the results of cocultures performed with nerve cells and normal or demedullated leg muscle myoblasts.