Comparison between adult and foetal adnexa derived equine post-natal mesenchymal stem cells.

BACKGROUND: Little is known about the differences among adult and foetal equine mesenchymal stem cells (MSCs), and no data exist about their comparative ultrastructural morphology. The aim of this study was to describe and compare characteristics, immune properties, and ultrastructural morphology of equine adult (bone marrow: BM, and adipose tissue: AT) and foetal adnexa derived (umbilical cord blood: UCB, and Wharton's jelly: WJ) MSCs. RESULTS: No differences were observed in proliferation during the first 3 passages. While migration ability was similar among cells, foetal MSCs showed a higher adhesion ability, forming smaller spheroids after hanging drop culture (P < 0.05). All MSCs differentiated toward adipogenic, chondrogenic and osteogenic lineages, only tenogenic differentiation was less evident for WJ-MSCs. Data obtained by PCR confirmed MHC1 expression and lack of MHC2 expression in all four cell types. Foetal adnexa MSCs were positive for genes specific for anti-inflammatory and angiogenic factors (IL6, IL8, ILß1) and WJ-MSCs were the only positive for OCT4 pluripotency gene. At immunofluorescence all cells expressed typical mesenchymal markers (α-SMA, N-cadherin), except for BM-MSCs, which did not express N-cadherin. By transmission electron microscopy, it was observed that WJ-MSCs had a higher (P < 0.05) number of microvesicles compared to adult MSCs, and UCB-MSCs showed more microvesicles than BM-MSCs (P < 0.05). AT-MSCs had a lower number of mitochondria than WJ-MSCs (P < 0.05), and mitochondrial area was higher for WJ-MSCs compared to UCB and AT-MSCs (P < 0.05). CONCLUSIONS: Results demonstrate that MSCs from adult and foetal tissues have different characteristics, and foetal MSCs, particularly WJ derived ones, seem to have some charactestics that warrant further investigation into potential advantages for clinical application.

Effects of mesenchymal stromal cells versus serum on tendon healing in a controlled experimental trial in an equine model.

BACKGROUND: Mesenchymal stromal cells (MSC) have shown promising results in the treatment of tendinopathy in equine medicine, making this therapeutic approach seem favorable for translation to human medicine. Having demonstrated that MSC engraft within the tendon lesions after local injection in an equine model, we hypothesized that they would improve tendon healing superior to serum injection alone. METHODS: Quadrilateral tendon lesions were induced in six horses by mechanical tissue disruption combined with collagenase application 3 weeks before treatment. Adipose-derived MSC suspended in serum or serum alone were then injected intralesionally. Clinical examinations, ultrasound and magnetic resonance imaging were performed over 24 weeks. Tendon biopsies for histological assessment were taken from the hindlimbs 3 weeks after treatment. Horses were sacrificed after 24 weeks and forelimb tendons were subjected to macroscopic and histological examination as well as analysis of musculoskeletal marker expression. RESULTS: Tendons injected with MSC showed a transient increase in inflammation and lesion size, as indicated by clinical and imaging parameters between week 3 and 6 (p < 0.05). Thereafter, symptoms decreased in both groups and, except that in MSC-treated tendons, mean lesion signal intensity as seen in T2w magnetic resonance imaging and cellularity as seen in the histology (p < 0.05) were lower, no major differences could be found at week 24. CONCLUSIONS: These data suggest that MSC have influenced the inflammatory reaction in a way not described in tendinopathy studies before. However, at the endpoint of the current study, 24 weeks after treatment, no distinct improvement was observed in MSC-treated tendons compared to the serum-injected controls. Future studies are necessary to elucidate whether and under which conditions MSC are beneficial for tendon healing before translation into human medicine.

[MR defaecography for the diagnosis of obstructive defaecation disorders]. / MR-Defäkografie bei obstruktiven Defäkationsstörungen.

As a consequence of demographic changes, outlet obstruction represents an increasingly common disease. The presence and ample interactions of morphological and functional pathologies contribute to the complexity of pelvic floor dysfunction. Additionally, multiple compartments of the pelvic floor are frequently affected. MR defaecography allows for the simultaneous and detailed assessment of morphological as well as functional changes of the pelvic floor. Hence, this approach constitutes an integral part of the diagnostic work-up and preoperative evaluation of the anorectum. The supine patient position can be regarded as a drawback compared to conventional defaecography, as sufficient emptying of the rectum can be impaired or even rendered impossible in individual cases. This inherent disadvantage is, however, compensated by the high anatomic resolution, the possibility of multiplanar imaging, easy execution and especially the lack of ionising radiation. Consequently, MR defaecography is considered the method of choice for the routine evaluation of functional anorectal disorders.

Scalable Production of Equine Platelet Lysate for Multipotent Mesenchymal Stromal Cell Culture.

Translation of multipotent mesenchymal stromal cell (MSC)-based therapies is advancing in human and veterinary medicine. One critical issue is the in vitro culture of MSC before clinical use. Using fetal bovine serum (FBS) as supplement to the basal medium is still the gold standard for cultivation of many cell types including equine MSC. Alternatives are being explored, with substantial success using platelet lysate-supplemented media for human MSC. However, progress lags behind in the veterinary field. The aim of this study was to establish a scalable protocol for equine platelet lysate (ePL) production and to test the ePL in equine MSC culture. Whole blood was harvested into blood collection bags from 20 healthy horses. After checking sample materials for pathogen contamination, samples from 19 animals were included. Platelet concentrates were prepared using a buffy coat method. Platelets, platelet-derived growth factor BB, and transforming growth factor ß1 concentrations were increased in the concentrates compared with whole blood or serum (p < 0.05), while white blood cells were reduced (p < 0.05). The concentrates were lysed using freeze/thaw cycles, which eliminated the cells while growth factor concentrations were maintained. Donor age negatively correlated with platelet and growth factor concentrations after processing (p < 0.05). Finally, all lysates were pooled and the ePL was evaluated as culture medium supplement in comparison with FBS, using adipose-derived MSC from four unrelated donor horses. MSC proliferated well in 10% FBS as well as in 10% ePL. However, using 5 or 2.5% ePL entailed highly inconsistent proliferation or loss of proliferation, with significant differences in generation times and confluencies (p < 0.05). MSC expressed the surface antigens CD90, CD44, and CD29, but CD73 and CD105 detection was low in all culture media. Adipogenic and osteogenic differentiation led to similar results in MSC from different culture media. The buffy coat method is useful to produce equine platelet concentrate with increased platelet and reduced white blood cell content in large scales. The ePL obtained supports MSC expansion similar as FBS when used at the same concentration (10%). Further investigations into equine MSC functionality in culture with ePL should follow.

'Say no': a feasibility trial of a brief intervention to reduce instances of indulgent energy-intake episodes.

The aim of this study is to examine the feasibility of a brief intervention to reduce instances of indulgent energy intake. Forty-five participants with a body mass index (BMI) ≥25 kg m-2 were randomized to one of three groups for 8 weeks. The control group was asked to complete a questionnaire every 4 days, the self-monitoring group was given the same instructions but also asked to 'say no' to indulgences. The self-monitoring and feedback group was asked to do the same but in addition to send a photograph or description of that to which they had 'said no' and were then provided with feedback. All participants reported on indulgences for 7 days prospectively at baseline and 8-week follow-up. The follow-up rate was 80%; completion of questionnaires was 63% and 87 text messages were sent. The control group reduced their indulgences by 4.1 (SD 10.0), the self-monitoring group by 13.8 (SD 16.8) and self-monitoring and feedback group by 9.0 (SD 11.7) per week. All bar one, feasibility progression criteria were met and this was the return of the indulgence diaries during the intervention period. The study demonstrates the feasibility of a brief intervention to reduce the number of indulgences people ate. The progression criteria were met and areas of improvement are highlighted.

Characterisation and intracellular labelling of mesenchymal stromal cells derived from synovial fluid of horses and sheep.

Multipotent mesenchymal stromal cells (MSCs) derived from synovial fluid (SF) are considered to be a promising cell type for therapeutic applications in joint disease. However, despite their potential relevance for clinical and experimental studies, there is insufficient knowledge about SF-derived MSCs isolated from horses and sheep. In this study, cells were recovered from healthy SF and bone marrow (BM) of sheep, and from healthy and osteoarthritic SF of horses. Ovine SF-MSCs were used to assess the efficiency of intracellular labelling with quantum dots (QDs). Colony forming units, generation times, trilineage differentiation potential and expression of CD73, CD90 and CD105 at mRNA level were assessed. QD labelling was efficient, with >98% positive cells directly after labelling at 10 nmol/L and >95% positive cells directly after labelling at 2 nmol/L. The label decreased over 7 days of culture, with more persistence at the higher labelling concentration. No significant differences in proliferation were observed. All MSCs had trilineage differentiation potential, but adipogenesis was more distinct in equine samples and chondrogenesis was most pronounced in ovine SF-MSCs. CD73, CD90 and CD105 were expressed in equine and ovine MSCs.

Parametrization of nanoparticles: development of full-particle nanodescriptors.

While metal oxide nanoparticles (NPs) are one of the most commonly used nanomaterials, the theoretical models used to analyze and predict their behavior have been mostly based on just the chemical composition or the extrapolation from small metal oxide clusters' calculations. In this study, a set of novel, theoretical full-particle descriptors for modeling, grouping or read-across of metal oxide NP properties and biological activity was developed based on the force-field calculation of the potential energies of whole NPs. The capability of these nanodescriptors to group the nanomaterials acoording to their biological activity was demonstrated by Principal Component Analysis (PCA). The grouping provided by the PCA approach was found to be in good accordance with the algal growth inhibition data of well characterized nanoparticles, synthesized and measured inside the consortia of the EU 7FP framework MODERN project.

Dissociation between the attentional functions mediated via basal forebrain cholinergic and GABAergic neurons.

The role of basal forebrain corticopetal cholinergic projections in attentional functions has been extensively investigated. For example, 192 IgG-saporin-induced loss of cortical cholinergic inputs was repeatedly demonstrated to result in a selective impairment in the ability of rats to detect signals in a task designed to assess sustained attention performance. The loss of cortical cholinergic inputs correlated highly with the decrease in the hit rate. Little is known about the functions of basal forebrain non-cholinergic neurons, particularly corticopetal GABAergic neurons, largely because of the absence of specific research tools to manipulate selectively this projection. As basal forebrain lesions produced with ibotenic acid were previously observed to potently destroy non-cholinergic, particularly GABAergic neurons while producing only moderate decreases in the density of cortical cholinergic inputs, the present experiment examined the effects of such lesions on sustained attention performance and then compared these effects with the immunohistochemical and attentional consequences of selective cholinotoxic lesions produced by intra-basal forebrain infusions of 192 IgG-saporin. In contrast to the selective decrease in hits previously observed in 192 IgG-saporin-lesioned animals, the attentional performance of ibotenic acid-lesioned animals was characterized by a selective increase in the relative number of false alarms, that is 'claims' for signals in non-signal trials. Analyses of the response latencies suggested that this effect of ibotenic acid was due to impairments in the animals' ability to switch from the processing of the response rules for signal trials to those for non-signal trials. As expected, 192 IgG-saporin did not affect the number of basal forebrain parvalbumin-positive neurons, that are presumably GABAergic, but decreased cortical acetylcholinesterase-positive fiber density by over 80%. Conversely, in ibotenic acid-lesioned animals, basal forebrain parvalbumin-positive cells were decreased by 60% but cortical acetylcholinesterase-positive fiber density was only moderately reduced (less than 25%). These data form the basis for the development of the hypothesis that basal forebrain GABAergic neurons mediate executive aspects of attentional task performance. Such a function may be mediated in parallel via basal forebrain GABAergic projections to the cortex and the subthalamic nucleus.

Differential cortical acetylcholine release in rats performing a sustained attention task versus behavioral control tasks that do not explicitly tax attention.

The present study used microdialysis techniques to compare acetylcholine release in the frontoparietal cortex of rats performing in a task requiring sustained attention with that of rats performing in two control procedures. The two control procedures were a fixed-interval 9-s schedule of reinforcement assessing primarily the effects of operant responding and comparable reward rates, and an operant procedure designed to test the effects of lever extension to prompt responding. These two control procedures involved comparable sensory-motor and motivational variables to those of the sustained attention task, but did not explicitly tax attentional processes. Performance of the sustained attention task was associated with a significant increase in cortical acetylcholine efflux, reaching a maximum of nearly 140%. Performance of the two control procedures was associated with significantly smaller (approximately 50%) increases in cortical acetylcholine release. This robust dissociation between attentional and control performance-associated increases in cortical acetylcholine release resulted, in part, from the elimination of the pre-task transfer of the animals into the operant chambers and the associated increases in acetylcholine release observed in previous studies. The present results support the hypothesis that demands on attentional performance, as opposed to the frequency of lever pressing, reward delivery and other task-related variables, selectively activate the basal forebrain corticopetal cholinergic system.

A sleep laboratory evaluation of an automatic positive airway pressure system for treatment of obstructive sleep apnea.

STUDY OBJECTIVES: This paper compares the performance of an experimental nasal positive airway pressure device that automatically adjusts the level of applied pressure (APAP) with the performance of a conventional continuous positive airway pressure (CPAP) in a sleep laboratory study. DESIGN: In a randomized sequence, conventional CPAP therapy was applied for 1 night (CPAP night) and APAP therapy the following night (APAP night). SETTING: The study was conducted in an accredited sleep disorders center. PATIENTS OR PARTICIPANTS: Twenty-six men and 5 women between the ages of 35 to 73 (51 +/- 9.6) years with body mass index 35.82 +/- 8.35 (kg/m2) who were diagnosed (using standard nocturnal polysomnography [NPSG] methods) as having OSA syndrome were studied. The subjects were treated with conventional CPAP for approximately 8 (7.79 +/- 3.16) weeks at home prior to their participation in this study. MEASUREMENTS AND RESULTS: All standard polysomnography data and nasal mask pressures were recorded using a computer-based data acquisition system. Sleep and respiratory data were scored by a registered polysomnographer. The mean apnea-hypopnea index (AHI) for subjects for the NPSG night was 55.2 +/- 33.7. It dropped to 4.2 +/- 3.8 for the CPAP night and to 5.4 +/- 5.4 for the APAP night. There was no significant (p = 0.05) difference between mean AHI indices, sleep stages, sleep stage shifts, and snore arousals for CPAP night and APAP night. However, all the measures showed significant (p = 0.05) improvement over NPSG night. The mean of APAP applied pressure (8.4 +/- 3.3 cm H2O) was significantly (p = 0.05) lower than the prescribed pressure (11.5 +/- 3.1 cm H2O), but there was no significant (p = 0.05) difference between the maximum APAP applied pressure (12.8 +/- 4.3 cm H2O) and the prescribed pressure (11.5 +/- 3.1 cm H2O). All mean comparison tests were carried out using two-tailed statistics. CONCLUSIONS: APAP appears to be as effective as CPAP in treating OSA patients. APAP delivers the same level of therapy as CPAP, but it reduces the average airway pressure while providing needed peak pressures.

Thalamic amnesia reconsidered: excitotoxic lesions of the intralaminar nuclei, but not the mediodorsal nucleus, disrupt place delayed matching-to-sample performance in rats (Rattus norvegicus).

Rats with large thalamic lesions affecting the mediodorsal (MDn) and intralaminar (ILn) nuclei are impaired performing delayed matching to sample tasks (DMTS). To determine the neurological basis of this deficit, we trained rats to perform a place DMTS task and then compared the effects of excitotoxic lesions of the MDn, the ILn, and the lateral internal medullary lamina (L-IML). The MDn lesion had little effect. The ILn group was significantly impaired throughout 8 months of training. The L-IML group exhibited an intermediate level of impairment. Varying the sample response requirement, retention intervals, and trial-to-trial congruence in the side reinforced, had predicted effects, as did variations in response latency. However, none of these factors interacted with the treatment effects. These findings indicate that DMTS performance is disrupted by lesions of the ILn but not the MDn.

Lesions of the frontal cortex, hippocampus, and intralaminar thalamic nuclei have distinct effects on remembering in rats.

Lesions of the intralaminar thalamic nuclei (ILn), the medial wall (MW) area of prefrontal cortex, and the hippocampus were compared and found to have distinct effects on delayed matching-to-sample (DMS) and delayed non-matching-to-sample (DNMS) tasks based on different types of stimulus cues. Hippocampal lesions impaired DNMS trained in a radial arm maze but had little effect on DMS trained with retractable levers or olfactory DNMS. MW lesions affected the DMS task but had limited effects on olfactory DNMS and radial arm maze DNMS. ILn lesions resulted in a more generalized pattern of impairment for radial maze tasks and (in previous studies) for the DMS and olfactory DNMS tasks. Only the hippocampal lesion was associated with a delay-dependent impairment. It is argued that ILn lesions disrupt remembering through their effects on the recurrent, feedback pathways that link functionally related areas of the basal ganglia and cortex.

Effects of thalamic and olfactory cortical lesions on continuous olfactory delayed nonmatching-to-sample and olfactory discrimination in rats (Rattus norvegicus).

We conducted 2 studies to determine the importance of several brain systems for remembering odorants in a go/no-go delayed nonmatching-to-sample (DNMTS) task. In Experiment 1, impairments were observed for lesions of pyriform cortex or (to a lesser extent) the lateral internal medullary lamina of thalamus. Lesions of the entorhinal cortex or the mediodorsal (MDn) or the paracentral and centrolateral (PC-CL) thalamic nuclei did not affect DNMTS. In Experiment 2, an impairment comparable to the pyriform lesion was observed for a lesion of the intralaminar nuclei (PC-CL plus the central medial nucleus) but not for a larger lesion of MDn. None of the lesions in either study affected the ability to learn a 2-choice odor discrimination using go/no-go procedures comparable with the DNMTS task.

Effects of chlordiazepoxide and FG 7142 on a rat model of diencephalic amnesia as measured by delayed-matching-to-sample performance.

The intralaminar thalamic nuclei (ILn) have been implicated as a critical site of pathology in amnesia. Lesions of the ILn have been found to produce behavioral effects comparable to benzodiazepine (BDZ) receptor agonists. We compared the effects of chlordiazepoxide (CDP), a BDZ agonist, and FG 7142, a partial inverse agonist at the BDZ receptor, in rats with thalamic lesions and in unlesioned controls. Delayed matching-to sample (DMS) performances were studied during treatment with ascending doses of CDP, counterbalanced trials with 2.5 mg/kg CDP and saline, ascending doses of FG 7142, and (for unlesioned controls only) counterbalanced trials with saline and higher doses of CDP. CDP had effects similar to the ILn lesion, decreasing response speed and percent correct responding in a delay-independent fashion. These effects were additive with the impairments associated with the ILn lesion. The effects of FG 7142 were more complex. At lower doses, it increased response speed without affecting response accuracy. At higher doses, it diminished both the speed and the accuracy of DMS responding. These results support the hypothesis that ILn lesions and BDZ agonists have similar effects on DMS performance. The biphasic effects observed for FG 7142 are consistent with other evidence that low doses of this drug enhance while higher doses impair memory performance. Although DMS accuracy was not improved, the enhancement observed for response speed provides evidence that partial inverse BDZ agonists have potential utility as treatments for cognitive impairments associated with amnesia.

Effects of dorsal and ventral striatal lesions on delayed matching trained with retractable levers.

Recent evidence has suggested that thalamic amnesia results from damage to the intralaminar nuclei, an important source of input to striatum. To test the hypothesis that intralaminar damage disrupts functions mediated by striatum, we studied the effects of striatal lesions on a delayed matching task known to be affected by intralaminar lesions. Rats were trained to perform the task and given one of five treatments: sham surgery or a lesion of medial or lateral caudate/putamen, nucleus accumbens, or ventral striatum. Rats with ventral striatal lesions were impaired compared to all other groups. Rats with medial caudate/putamen or nucleus accumbens lesions were impaired compared to controls. The effects of ventral striatal lesions were sufficient to account for impairments in the accuracy and latency of delayed matching responses observed in previous studies of intralaminar and medial frontal cortical lesions. The ventral striatal lesions involved portions of ventral pallidum and thus it seems likely that they affected functions mediated by the nucleus accumbens as well as striatal areas of the tubercle. Serial reversal learning trained in the same apparatus with the same reinforcer was unaffected by all of the lesions. These results are discussed in terms of the roles of midline thalamic nuclei and of thalamo-cortico-striatal circuits in delayed conditional discrimination tasks.

Effects of intralaminar thalamic lesions on sensory attention and motor intention in the rat: a comparison with lesions involving frontal cortex and hippocampus.

A self-paced serial reaction task was developed to differentiate between the effects of intralaminar thalamic lesions on sensory attention and intentional motor function. Results were compared for hippocampal and frontal cortical lesions to test for the possible involvement of pathways involving these parts of the brain in any impairments associated with the thalamic lesion. Lesions of the intralaminar thalamic nuclei affected response latency without affecting accuracy. This increase in latency was unaffected by variations in stimulus duration, even though this manipulation had a substantial effect on response accuracy. Intralaminar lesions did not affect the response to distracting stimuli or to manipulations of stimulus salience. Thus it seems unlikely that the effects of intralaminar lesions on motor function were related to sensory loss or attentional dysfunction. Hippocampal lesions had no significant effect on any measure of performance. Frontal cortical lesions were associated with an increase in latency comparable to the intralaminar group and also affected the accuracy of responding to brief stimuli or under conditions of reduced stimulus salience. These results are discussed in light of evidence that lesions of the intralaminar nuclei affect functions mediated by anatomically related areas of frontal cortex and striatum.

A comparison of the effects of hippocampal or prefrontal cortical lesions on three versions of delayed non-matching-to-sample based on positional or spatial cues.

We trained rats to perform one of three versions of delayed non-matching-to-sample (DNMS): DNMS between two retractable levers in an enclosed operant chamber; varying-choice DNMS between two arms selected at random on every trial in an uncovered eight-arm radial arm maze; or recurring-choice DNMS between the same two arms on every trial in a covered radial maze (N=33/task). Rats with medial prefrontal cortical lesions showed delay-independent impairments on the retractable lever and recurring-choice tasks, but performed varying-choice DNMS normally. Rats with hippocampal lesions exhibited delay-independent impairments of the retractable lever task and delay-dependent impairments of both radial maze tasks. When rats trained initially to perform recurring choice DNMS were switched to varying choice DNMS, the impairments of both the prefrontal and hippocampal groups were reduced, although hippocampal animals remained significantly impaired. When rats trained initially to perform varying choice DNMS were switched to recurring choice DNMS, the impairment of the hippocampal group was exacerbated while the prefrontal group remained unimpaired. Thus training the prefrontal group to perform the varying choice task first seemed to protect from impairment when these rats were subsequently trained to perform recurring choice DNMS. This protection provides evidence against the possibility that factors related to proactive interference or to temporal discrimination can account for the effects of prefrontal lesions on delayed conditional discriminations involving two response alternatives in fixed locations.

Resource allocation patterns of two California-Sonoran desert ephemerals.

The patterns of allocation of structural and nonstructural carbon were followed in the co-occurring desert ephemerals Plantago insularis and Camissonia boothii. Patterns of biomass distribution were determined from material harvested at biweekly intervals as were levels of nonstructural sugar and starch. Seasonal patterns of growth and reproduction differed markedly with Plantago allocating significantly more structural and nonstructural carbon to reproduction early in the season. Plantago completed its life cycle in less than 60 days but Camissonia continued both vegetative and reproductive growth to over 100 days. The longer growing season of Camissonia was possible because more energy was allocated to vegetative tissues and storage presumably as investment toward longer life and higher levels of reproduction.

Differential effect of subtype of parental major affective disorder on children.

Fifty offspring of patients with major affective disorder (MAD) were studied. The children and their parents were interviewed using a structured diagnostic instrument. The results supported previous studies that the children of patients with MAD endorse a variety of symptoms of psychopathology. In addition, when the sample was separated by subtype of parental MAD, few differences in the types of symptoms were reported, and the differences that were found may be spurious. The differences, as well as indications for future research, are discussed.

Avascular necrosis of the femoral head: early MRI detection and radiological correlation.

Magnetic Resonance Imaging (MRI) and conventional radiographs were compared in 49 hips with Avascular Necrosis (AVN). MRI detected AVN in 25% of the hips during the preradiological stage of the disease. Both MRI and conventional radiographs accurately detected AVN in the remaining 75% of hips. Correlation between the patterns observed with the two techniques reflected the underlying histopathologic events. The reactive interface between infarcted bone and viable bone could be identified on MRI as a low signal intensity (SI) band. On conventional radiographs the reactive interface appeared as a sclerotic band. The adjacent hyperemic zone was seen on MRI as a high SI band and as a lucent zone on the plain films. Variations of this pattern occurred as related to the extend and duration of AVN and to the individual's ability to mount a healing response. Minor degrees of collapse of the femoral head were better identified with plain radiographs but MRI demonstrated small areas of hyperintensity probably corresponding to early subchondral fractures.