Biocompatibility of alendronate-loaded acrylic cement for vertebroplasty.

This paper reports a biological evaluation of a non-resorbable acrylic cement loaded with alendronate for the treatment of osteoporotic vertebral compression fractures. The cement formulation was based on polymethyl methacrylate and acrylic monomers; one of these had covalently linked vitamin E residues. The same cement in the absence of alendronate was used as a control. The setting of the charged cement presented a maximum polymerization temperature of 44°C, a setting time of 24 min, a residual monomer content lower than 3 wt.%, a compressive strength of 99±10 MPa and an elastic modulus of 1.2±0.2 GPa. Cytotoxicity studies using human osteoblast cultures revealed that the leachable substances of the alendronate loaded cement collected between 1 and 7 days decreased cell viability to values lower than 80%. However, morphological changes and cellular damage in cells produced by the extracts decreased with the leak time. Cell adhesion and growth on charged cement was significantly lower than on the control. Implantation of the cement paste in the intra-femoral cavity of rabbits showed that initially the osteogenic activity was evident for the cement charged with alendronate, and the osteosynthesis process took place mainly in the trabeculae and was manifested by the presence of a non-mineralised osseous spicule. The interface between material and adjacent bone tissue was initially characterized by a variable fibrous response that in many cases it appeared reduced to thin connective tissue after a 24-week-period.

Injectable self-curing bioactive acrylic-glass composites charged with specific anti-inflammatory/analgesic agent.

Injectable bioactive acrylic formulations based on poly(methyl methacrylate) (PMMA) and different amounts of bioactive glasses in the system SiO2-CaO-Na2O-P2O5 have been prepared in the presence of the anti-inflammatory analgesic drug fosfosal, the sodium salt of 2-phosphonoxibenzoic acid, to be used in minimally invasive surgery. The injectability of the formulations evaluated according to the established protocol was around 80%. The experimental formulations provided maximum temperatures in the range 50-60 degrees C, which were lower than those of commercial acrylic bone cements currently used in percutaneous vertebroplasty (PVP). Residual monomer content of any formulation was inferior to 5%. Compressive yield strength of dry specimens was in the range 80-95 MPa, but it decreased after immersion in SBF to values in the range 30-50 MPa, due to the dissolution of the bioactive glasses and the drug in the medium. The release of fosfosal was evaluated in vitro (pH = 7.0). The release profile against time obtained from a PMMA cement was quasi-linear and the 80% of the initial amount of drug was released in 175 h. However, for bioactive cements, the 80-100% of the fosfosal charged was released in approximately 48 h, due to the dissolution of the glasses in the medium. Values of weight loss of the cements determined gravimetrically ranged between 16% and 26% depending on the initial amount of fosfosal, i.e. 20 or 30 wt%, respectively. The weight loss and the water uptake were simultaneous processes, and values of hydration degree were around 10-14%. The formation of an apatite-like layer was detected on the surface of the cements at different periods of time depending on the composition of the bioactive glasses. The cements containing the glasses with P2O5 produced the growth of the apatite layer in shorter periods of time. The presence of fosfosal accelerated the precipitation of this layer independently on the glasses. The in vivo biocompatibility studied by intramuscular implantation in rats showed the absence of an anti-inflammatory response and a fibrous layer around the implant for the cement prepared with PMMA/fosfosal which is attributed to the therapeutic action of fosfosal acting in situ. The response to cements prepared with bioactive glasses and fosfosal showed a mild inflammatory reaction with the formation of the typical fibrous capsule around the implanted material.

Biocompatibility and other properties of acrylic bone cements prepared with antiseptic activators.

Acrylic bone cements prepared with activators of reduced toxicity have been formulated with the aim of improving the biocompatibility of the final material. The activators used were N,N-dimethylaminobenzyl alcohol (DMOH) and 4,4'-dimethylamino benzydrol (BZN). The toxicity, cytotoxicity, and antiseptic action of these activators were first studied. DMOH and BZN presented LD50 values 3-4 times higher than DMT, were less cytotoxic against polymorphonuclear leucocytes, and possessed an antimicrobial character, with a high activity against the most representative microorganisms involved in postoperative infections. The properties of the acrylic bone cements formulated with DMOH and BZN were evaluated to determine the influence of these activators on the curing process and the physicochemical characteristics of the cements. A decrease of the peak temperature was observed for the curing with DMOH or BZN with respect to that of one commercially available formulation (CMW 3). However, residual monomer content and mechanical properties in tension and compression were comparable to those of CMW 3. The biocompatibility of acrylic bone cements containing DMOH or BZN was studied and compared with CMW 3. To that end, intramuscular and intraosseous implantation procedures were carried out and the results were obtained from the histological analysis of the surrounding tissues at different periods of time. Implantation of rods of cement into the dorsal muscle of rats showed the presence of a membrane of connective tissue, which increased in collagen fibers with time of implantation, for all formulations. The intraosseous implantation of the cements in the dough state in the femur of rabbits, revealed a higher and early osseous neoformation, with the presence of osteoid material surrounding the rest of the cured material, for the cement prepared with the activator BZN in comparison with that obtained following the implantation of the cement cured with DMOH or DMT (CMW 3).

Projections from the primary auditory cortex onto the dorsal cortex of the inferior colliculus in albino rats.

The topography of connections from the primary auditory cortex (Te1) onto the dorsal cortex (DC) of the inferior colliculus (IC) was studied using the anterograde neural tracers Phaseolus vulgaris (PHA-L) and Biotinylated dextran amine (BDA). Injections in different restricted portions of the ventral Te1 showed labelled axonal sheets in the ipsilateral DC with extensions in the central nucleus and the external cortex. Also contralateral labelled axons were found with a patchy appearance. The pattern of labelled axonal laminae is arranged in an orderly fashion, so that a correlation exists between the injection field (antero-to caudoventral injections) and the pattern of labelled axonal sheets (from a ventromedial to a dorsolateral arrangement) in the IC. However, variations from this pattern were observed. Also only the injections made in rostral and middle parts of Te1 V are orderly arranged in the territory of the DC, while the Te1 V caudoventral projection partially overlaps those from antero and mid-central regions. A topographical arrangement in the fibre projection from the medial and dorsal zones of Te1 (Te1 D) exists in the neuropil of the IC. The bands from Te1 D overlapped with the bands from Te1 V but in different order. This indicates that the projection of the mid-dorsal zone of Te1 is not topographically arranged in the DC like Te1 V. Based on the results we have obtained, there is strong evidence that the projection from Te1 V to the DC is topographically organized in a rostrocaudal sequence which gives definite terminal fields. However, each of these fields (medial, central and external) receives a principal bulk of primary auditory cortical fibres and a supplementary one. This implies that territories of the DC which contain neurons responsive to low frequencies receive a projection from Te1 fields with neurons which are responsive to high or to low frequencies.

Biological response of new activated acrylic bone cements with antiseptic properties. Histomorphometric analysis.

The biological response to an acrylic bone cement cured with 4,4'-bis-dimethylamino benzydrol (BZN) as activator of reduced cytotoxicity and antiseptic properties, has been carried out and compared with that obtained for CMW 3 cement. Histomorphometrical data (undecalcified trichromic Goldner staining) were obtained by measuring the most significant variables at the bone-cement interface. Quantitative results of tissue response revealed that newly formed bone and connective tissue were maximum at 4 weeks whereas bone marrow increased with time of implantation for both cements. Statistical analysis (p < 0.05) showed no significant differences in newly formed bone and bone marrow with time and between both groups, however, connective tissue significantly decreased between 4 weeks and 12 weeks for BZN cement, and between 12 weeks and 24 weeks for CMW3. By comparing both cements at each time, lower significant percentage of connective tissue at the bone-cement interface of the BZN cement, was obtained at 12 and 24 weeks, however, a very low amount of connective tissue was found for both cements. All the results indicate that the new activated system could be applied clinically in a relatively short time, after the corresponding preclinical study.

Histomorphometric study of femoral heads in hip osteoarthritis and osteoporosis.

During the period from 2000 to 2003, ninety eight samples of femoral heads were collected. In these pieces, two zones were analyzed: a high-load zone (the hard core of the head) and a low-load zone (the round ligamentum teres zone). As control group, 6 femoral heads (3 of women and 3 of men), proceeding from autopsy in peoples without pathological antecedents and youngs, were studied. After the samples had been embedded in methylmethacrylate and stained, they were subjected to an histomorphometric study. By means of histomorphometry, trabecular bone volume (TBV) and osteoid substance (OSV) was determined. Statistically significant differences were found as for peripheral osteoid volume (low-load zone) (p=0.036) and trabecular bone volume, both peripheral and central. Both volumes decreased in osteoporotic samples and in those from women (p=0.000), in comparison with control group. Regarding the relationship between the high-load and low-load zone, significant data were obtained. The high-load zone had a greater trabecular bone volume than the low-load zone, regardless of the pathology and sex, but this increase was more pronounced in the arthrosic samples and in those from men. Additionally, this trabecular bone volume in the high-load zone decreased with increasing age of the donor (p=0.037), when the control group is compared. In sum, we observed a reduction in the formation of TBV and OSV in osteoporosis but also a decrease in the arthrosic, in samples from older subjects, in women, and in the low-load zone of the samples, when the control group is compared. These data suggest the coexistence of both pathologies, which is more pronounced in older subjects and women.

Pulsed electromagnetic fields induce peripheral nerve regeneration and endplate enzymatic changes.

An experimental study was carried out in rats with the purpose of demonstrating the capacity of pulsed electromagnetic fields (PEMFs) to stimulate regeneration of the peripheral nervous system (PNS). Wistar and Brown Norway (BN) rats were used. Direct sciatic nerve anastomoses were performed after section or allograft interposition. Treatment groups then received 4 weeks of PEMFs. Control groups received no stimulation. The evaluation of the results was carried out by quantitative morphometric analysis, demonstrating a statistically significant increase in regeneration indices (P < 0.05) in the stimulated groups (9000 +/- 5000 and 4000 +/- 6000) compared to the non-stimulated groups (2000 +/- 4000 and 700 +/- 200). An increase of NAD specific isocitrate dehydrogenase (IDH) activity was found along with an increase in the activity of acetyl cholinesterase at the motor plate. The present study might lead to the search for new alternatives in the stimulation of axonal regenerative processes in the PNS and other possible clinical applications.

Untoward effects of pharmacological doses of insulin in early chick embryos: through which receptor are they mediated?

The teratogenic effect of insulin in early vertebrate embryos is controversial and the mechanisms involved are unknown. We studied the effects of pharmacological doses of insulin in chick embryos during the period of differentiation. We compared the effects of insulin with two proinsulins, desoctapeptide-insulin and multiplication-stimulating activity, peptides that have little insulin-like metabolic activity while they have significant growth effects. Chick embryos at 46 h of development were injected with the different peptides. At 96 h the mortality and abnormal growth elicited by the peptides were dose-dependent. Considering the indices of lethality (LD50) and affected embryos (ED50) as 100% for insulin, proinsulin was 59-66% as potent as insulin, desoctapeptide-insulin 2-6% and multiplication-stimulating activity 176-204%. In the surviving embryos, insulin (5 micrograms, decreased DNA, RNA and protein content by 49%, 40% and 48% respectively compared with controls. The effects of insulin were not corrected by simultaneous glucose injections. These data suggest that insulin, at pharmacological doses, interferes with embryo development through a non-metabolic pathway, probably via a growth-type receptor.

Morphology of cochlear root neurons in the rat.

The morphology of large neurons in the cochlear nerve root of albino rat has been studied with a variety of techniques including Nissl and cell-myelin staining, Golgi impregnation, horseradish peroxidase back-filling of severed axons, transmission electron microscopy, and morphometry. The cells, called root neurons, resemble the globular cells of the ventral cochlear nucleus in having an oval cell body, an eccentric nucleus, an axon that projects centrally via the trapezoid body, and in receiving many primary-like axosomatic boutons. The root neurons, however, are larger than globular cells, and they have at least two types of dendrites oriented, respectively, parallel and across the cochlear nerve fibres. The soma, moreover, has less finely dispersed Nissl material, is less completely covered with terminals, and receives a smaller proportion of presumably inhibitory synapses. So far, this particular type of neuron has been observed only in rat and mouse.

Morphology of the primary posterior plexus of the rat cochlear nucleus.

Golgi impregnation results confirm the existence of a plexus (previously described with horseradish peroxidase tracing techniques) located in the posteroventral cochlear nucleus. The bulk of axons involved in this plexus could be traced to their origin in the root, and were not related to the V-bifurcating fibres, though some of the posterior branches of the latter contributed to the plexus. The internal structure of the plexus, and the different types of endings are described. These findings are discussed in relation to previous Golgi studies, especially those referring to the neuron and axon population of this area.

Distribution of primary cochlear afferents in the bulbar nuclei of the rat: a horseradish peroxidase (HRP) study in parasagittal sections.

HRP was injected into the cochleae of 25 young albino rats in order to trace the primary afferents to the bulbar cochlear nuclei. Besides the classic V-shaped pattern and unconnected with it, HRP labelling revealed two plexuses stemming directly from the axons of the cochlear root. The plexuses cover the posterior area of the posteroventral cochlear nucleus (posterior plexus) and the anterolaterodorsal area of the anteroventral cochlear nucleus (anterior plexus). The fibres giving rise to these two plexuses were previously grouped in two bundles which have been called the posterior and anterior bundles, respectively. The origin of the anterior bundle is typically seen with the fibres stemming out at right angles; the origin and course of the posterior bundle, which characteristically cross over, is also a typical feature.

Distribution of primary afferent fibres in the cochlear nuclei. A silver and horseradish peroxidase (HRP) study.

Horseradish peroxidase, when injected intracochlearly, is transported transganglionically to the brain stem cochlear nuclei, thus providing an excellent method for tracing the central projection of the spiral ganglion neurons. Silver impregnation using the Cajal-de Castro method, which stains axons even when inside the bone, was used as a reference technique. The combination of both procedures led to the following conclusions. Primary cochlear afferents are found only in the ventral zone of the dorsal cochlear nucleus. In this area they cover the deep and fusiform cell layers. The molecular layer shows no HRP label. The higher concentration of primary cochlear afferents in the ventral cochlear nucleus appears in its central zone; wide areas in this nucleus are not labelled at all. A thin bundle of primary cochlear afferents runs parallel to, and beneath, the granular region.

Prenatal intramedullary gliofibroma. A light and electron microscope study.

A case of an unusual congenital intramedullary tumor of the spinal cord is reported. A paraplegic 11-day-old boy with hypotonia and atrophy of the abdominal and lower-extremity muscles showed a complete myelographic block between T-5 and T-8. Surgical exploration disclosed an elongated tumor mass within the spinal cord, that blended with the surrounding nervous-system tissue. Light and electron microscopy showed that the tumor was composed of intermingled well differentiated astrocytes and fibroblasts. These two cell types often were surrounded by the same basal lamina. There were no intercellular junctions. Gliofibrils were abundant, and the interstitial spaces contained abundant collagen and reticulin fibers. There were no histological signs of malignancy. We conclude that this is a case of prenatally arising gliofibroma.

Cytogenetic study of spontaneous abortions by transabdominal villus sampling and direct analysis of villi.

We report our experience in a cytogenetic study of 93 spontaneous abortions. Specimens were obtained by transabdominal chorionic villus sampling (TACVS) in women requesting prenatal diagnosis by chorionic villus sampling (CVS) but in whom an arrested pregnancy had been diagnosed during the ultrasound examination. Our success rate, i.e. the percentage of cases where we obtained results, was 91. 4 per cent, and the rate of abnormalities-mostly aneuploidies and polyploidies-was 62.3 per cent. In normal cases, masculine:feminine ratio was 1:1. These results confirm those obtained by other groups earlier this decade and allow us to conclude that, for the cytogenetic study of spontaneous abortions, CVS is a better approach than the culture of the products of conception after evacuation, because the success rate is higher and because it provides certainty that the specimens obtained are of fetal origin.

Local pathological findings in experimental dorsal root entry zone lesions performed by mechanical section, laser and radiofrequency. A comparative study.

Dorsal root entry zone lesions are nowadays accepted as a first choice of treatment to alleviate certain types of chronic pain though it is still controversial to decide proper procedure for lesioning. Based on this last argument in 15 mongrel dogs different types of lesions using mechanical microsection, radiofrequency and laser were done. Under general anaesthesia a C-3 to C-7 laminectomy was performed in all animals and after open the dura mater the posterior nerve rootlets and posterolateral sulcus were identified using magnification. In 5 dogs, a longitudinal 1 mm deep incision from C-4 to C-6 spinal cord segments coinciding with the sulcus was carried out with a microknife; in other 5, a sequence of thermocoagulations produced by a radiofrequency current lower than 35 mA was done at same places and level with intervals of 2 mm; and in the rest of animals, an alike incision in depth, location and level was realised by carbon dioxide laser. Four days and three months after lesioning 2 and 3 animals of each group were sacrificed and spinal cord specimens submitted for histological and ultrastructural studies. Low-power microscopic examination showed that all lesioning methods were able to produce a reasonable well delimited necrotic area involving the whole dorsal root entry zone structures, slightly more diffuse with the laser. Acute perilesional changes as well as chronic ones were nevertheless more evident in mechanical and radiofrequency lesions, over all regarding oedema, perivascular haemorrhage, intraluminal thrombosis and ischaemic alterations.