A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 μm in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 μm below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.     

Earliest Cranio-Encephalic Trauma from the Levantine Middle Palaeolithic: 3D Reappraisal of the Qafzeh 11 Skull,Consequences of Pediatric Brain Damage on Individual Life Condition and Social Care

The Qafzeh site (Lower Galilee, Israel) has yielded the largest Levantine hominin collection from Middle Palaeolithic layers which were dated to circa 90–100 kyrs BP or to marine isotope stage 5b–c. Within the hominin sample, Qafzeh 11, circa 12–13 yrs old at death, presents a skull lesion previously attributed to a healed trauma. Three dimensional imaging methods allowed us to better explore this lesion which appeared as being a frontal bone depressed fracture, associated with brain damage. Furthermore the endocranial volume, smaller than expected for dental age, supports the hypothesis of a growth delay due to traumatic brain injury. This trauma did not affect the typical human brain morphology pattern of the right frontal and left occipital petalia. It is highly probable that this young individual suffered from personality and neurological troubles directly related to focal cerebral damage. Interestingly this young individual benefited of a unique funerary practice among the south-western Asian burials dated to Middle Palaeolithic.     

Spag17 Deficiency Results in Skeletal Malformations and Bone Abnormalities

Height is the result of many growth and development processes. Most of the genes associated with height are known to play a role in skeletal development. Single-nucleotide polymorphisms in the SPAG17 gene have been associated with human height. However, it is not clear how this gene influences linear growth. Here we show that a targeted mutation in Spag17 leads to skeletal malformations. Hind limb length in mutants was significantly shorter than in wild-type mice. Studies revealed differences in maturation of femur and tibia suggesting alterations in limb patterning. Morphometric studies showed increased bone formation evidenced by increased trabecular bone area and the ratio of bone area to total area, leading to reductions in the ratio of marrow area/total area in the femur. Micro-CTs and von Kossa staining demonstrated increased mineral in the femur. Moreover, osteocalcin and osterix were more highly expressed in mutant mice than in wild-type mice femurs. These data suggest that femur bone shortening may be due to premature ossification. On the other hand, tibias appear to be shorter due to a delay in cartilage and bone development. Morphometric studies showed reduction in growth plate and bone formation. These defects did not affect bone mineralization, although the volume of primary bone and levels of osteocalcin and osterix were higher. Other skeletal malformations were observed including fused sternebrae, reduced mineralization in the skull, medial and metacarpal phalanges. Primary cilia from chondrocytes, osteoblasts, and embryonic fibroblasts (MEFs) isolated from knockout mice were shorter and fewer cells had primary cilia in comparison to cells from wild-type mice. In addition, Spag17 knockdown in wild-type MEFs by Spag17 siRNA duplex reproduced the shorter primary cilia phenotype. Our findings disclosed unexpected functions for Spag17 in the regulation of skeletal growth and mineralization, perhaps because of its role in primary cilia of chondrocytes and osteoblasts.     

2 trephined skulls from Schleswig-Holstein

In Schleswig-Holstein two trephined skulls are found, one belonging to a man deceased in the first half of adult age, who was buried in a stone grave of Middle Neolithic Age from Nebel on the isle of Amrum (published by Schaefer 1958, 1961). Another well preserved one, but without known site in Schleswig-Holstein, is the calvarium of a young adult presumably male showing a circular trephination without any tendency of healing. There is no symptom of a pathological change at the inner vault of the skull, but for the coronal suture gap . Striking are the grooves at both sides of the frontal bone which are quite unknown in this country. They are sulci arteriosi or nervi as is proved by Spanish skulls from Santa Cruz de Teneriffe and cases presented by Dixon (1904). EPIGENETIC TRAITS: 1. One sutural ossicle. 2. Incisura orbitalis r. 3. Threefold foramen supraorbitale 1. 4. Sulci arteriosi on the frontal bone. 5. Aplasia of left right molar (X-ray). PATHOLOGICAL CHANGES: 1. Hypoplasia of frontal sinuses. 2. Closing alveoli of first right molar. The case of a skull from Bremen (Domsdüne) demonstrates by the applied tool, that there are also trephinations in the Middle Neolithic Age.     

Free tissue coverage of chronic traumatic wounds of the lower leg

Thirty-eight consecutive patients who underwent 42 free flaps for chronic wounds of the lower leg were identified over an 11-year period. All wounds were open for a minimum of 1 month (mean, 40 months; median, 8 months; range, 1 month to 30 years). The average age was 37 years (range, 7 to 68 years), there were 31 male patients and seven female patients, and the average follow-up time was 30 months (range, 12 to 72 months). The original injury was an open fracture in 28 patients, wound dehiscence after open reduction and internal fixation of a closed fracture in nine patients, and a shrapnel wound in one patient. A total of 23 patients had osteomyelitis, which was classified as local (involving less than 50 percent of the bone diameter) in 15 patients and as diffuse (involving greater than 50 percent of the bone diameter or infected nonunion) in eight patients. The wounds were treated with sequential debridement, antibiotics, and flap coverage. Ancillary procedures included antibiotic beads in 18 patients, saucerization in 16, Ilizarov bone transport in three, calcanectomy in two, and fibular resection and ankle fusion in one. Thirty-four of 42 flaps survived, four having undergone a repeat free flap. There were three failures out of 25 flaps (12 percent) among those with a normal angiogram and five failures out of 15 flaps (33 percent) among those with an abnormal angiogram (p > 0.05). The failure rate of those with osteomyelitis was six of 26 (23 percent) versus two of 26 (13 percent) for those without osteomyelitis (p > 0.05). Successful reconstruction (bone healed, patient ambulatory and infection-free) was achieved in 33 of 38 patients (87 percent). The failure of reconstruction for those patients with osteomyelitis was four of 23 (22 percent) versus one of 15 (7 percent) for others (p > 0.05). The failure rate of flaps in patients with diffuse osteomyelitis was three of eight (38 percent) versus two of 30 for others (7 percent, p = 0.053). The presence of diffuse osteomyelitis was associated with a lower rate of successful limb reconstruction. An abnormal angiogram and the presence of osteomyelitis both were associated with a lower rate of successful limb reconstruction, but this was not significant, probably because of the small size of the cohort.     

Chronic osteomyelitis due to Clostridium difficile.

Osteomyelitis caused by anaerobic bacteria is rarely reported, and a case of chronic osteomyelitis of the femur may be the first in which Clostridium difficile was the causative agent. The organism was isolated over several months and, although initially sensitive to penicillin, it developed resistance during this time. The organism''s repeated isolation may have been due to the presence of resistant spores. Although the patient had no gastrointestinal symptoms the source of the organism was probably the patient''s own gastrointestinal tract. Infection from the environment cannot, however, be excluded. Treatment was finally successful with metronidazole.     

The simple and effective choice for treatment of chronic calcaneal osteomyelitis: neurocutaneous flaps

The authors describe their experience with the use of distally based saphenous and sural neurofasciocutaneous flaps for the treatment of calcaneal osteomyelitis in nine cases. Aggressive débridement of all nonviable and poorly vascularized tissue and coverage with a distally based neurofasciocutaneous flap were coupled with a thorough antibiotic course in all cases. The deepithelized peripheral parts of all flaps were buried in the bone cavities after bone débridement. Follow-up periods ranged from 15 to 27 months. All flaps survived completely. All of the wounds except one healed completely. These flaps have adequate blood flow for the management of chronic bone infections. They also have many advantages, such as easy quick elevation, short operative time, and acceptable donor-site morbidity. Moreover, patients treated with neurocutaneous flaps do not require debulking procedures or special shoes. Reconstruction with neurocutaneous flaps after radical débridement is a versatile alternative to the use of local or distant muscle flaps and calcanectomy procedures for patients with osteomyelitis of the os calcis.     

In vivo efficacy of tobramycin-loaded synthetic calcium phosphate beads in a rabbit model of staphylococcal osteomyelitis

Background

Osteomyelitis is an acute or chronic inflammatory process of the bone following infection with pyogenic organisms like Staphylococcus aureus. Tobramycin (TOB) is a promising aminoglycoside antibiotic used to treat various bacterial infections, including S. aureus. The aim of this study was to investigate the efficacy of tobramycin-loaded calcium phosphate beads (CPB) in a rabbit osteomyelitis model.

Methods

Tobramycin (30 mg/mL) was incorporated into CPB by dipping method and the efficacy of TOB-loaded CPB was studied in a rabbit osteomyelitis model. For juxtaposition, CPB with and without TOB were prepared. Twenty-five New Zealand white rabbits were grouped (n?=?5) as sham (group 1), TOB-loaded CPB without S. aureus (group 2), S. aureus only (group 3), S. aureus?+?CPB (group 4), and S. aureus?+?TOB-loaded CPB (group 5). Groups infected with S. aureus followed by CPB implantation were immediately subjected to surgery at the mid-shaft of the tibia. After 28 days post-surgery, all rabbits were euthanized and the presence or absence of chronic osteomyelitis and the extent of architectural destruction of the bone were assessed by radiology, bacteriology and histological studies.

Results

Tobramycin-loaded CPB group potentially inhibited the growth of S. aureus causing 3.2 to 3.4 log₁₀ reductions in CFU/g of bone tissue compared to the controls. Untreated groups infected with S. aureus showed signs of chronic osteomyelitis with abundant bacterial growth and alterations in bone architecture. The sham group and TOB-loaded CPB group showed no evidence of bacterial growth.

Conclusions

TOB-incorporated into CPB for local bone administration was proven to be more successful in increasing the efficacy of TOB in this rabbit osteomyelitis model and hence could represent a good alternative to other formulations used in the treatment of osteomyelitis.

     

Septic arthritis and osteomyelitis from a cat bite

A 39-year-old man with no prior history of underlying arthritis developed osteomyelitis and septic arthritis in his hand following a cat bite. This case illustrates the virulence of Pasteurella multocida infections associated with animal bites, particularly those of cats, whose teeth can inoculate bone directly. The onset of cellulitis caused by P. multocida infections is often rapid, and the drug of choice for such infections remains penicillin. Appropriate antibiotic therapy, however, does not always prevent complications such as those seen in this patient.     

A review of age determination methods for the Stoat Mustela erminea

A sample of 166 Stoats collected from Craigieburn Forest Park, Canterbury, New Zealand, was used to assess the usefulness of seven different methods of age determination. All the methods made use of characters which do change with age, but not all are equally good at defining useful age classes. The recommended approach is to use a combination of skull and baculum measurements to identify young animals, followed by counting of the canine cementum annuli of adults. If the skull or baculum is broken or not available, visual assessment of the status of the nasal sutures, the lateral suprasesamoid tubercle of the femur and the wear of the carnassial teeth are the next best options for distinguishing the young. Kopein's index based on the closure of the pulp cavity of the canines, and the zonation visible in the periosteal bone of the mandible, are unreliable.     

Possible osteomyelitis of skull causes death of a wild lowland gorilla in the Lopé Reserve, Gabon.

The remains of a juvenile gorilla, apparently dead for 3-4 days, were found in the Lopé Reserve, Gabon. The skull was recovered and showed a chronic lesion involving the insertion of the masseter muscle, consistent with chronic osteomyelitis.     

High-fat diet disrupts bone remodeling by inducing local and systemic alterations

A high-fat (HF) diet leads to detrimental effects on alveolar bone (AB); however, the mechanisms linking adiposity to bone loss are poorly understood. This study investigated if AB resorption induced by an HF diet is associated with the regulation of inflammatory gene expression and if adipocytes can directly interfere with osteoclastogenesis. We also evaluated the effects of diet restriction (DR) on bone phenotype. C57BL6/J mice were fed normal chow or an HF diet for 12 weeks. Samples of maxillae, femur, blood and white adipose tissue were analyzed. In vitro co-culture of bone marrow-derived osteoclasts and mature adipocytes was carried out. The results revealed an increased number of osteoclasts and fewer osteoblasts in animals fed the HF diet, which led to the disruption of trabecular bone and horizontal AB loss. Similar effects were observed in the femur. The metabolic parameters and the deleterious effects of the HF diet on AB and the femur were reversed after DR. The HF diet modulated the expression of 30 inflammatory genes in AB such as Fam3c, InhBa, Tnfs11, Ackr2, Pxmp2 and Chil3, which are related to the inflammatory response and bone remodeling. In vitro, mature adipocytes produced increased levels of adipokines, and co-culture with osteoclasts resulted in augmented osteoclastogenesis. The results indicate that the mechanisms by which an HF diet affects bone involve induction of osteoclastogenesis and inflammatory gene expression. Adipokines apparently are key molecules in this process. Strategies to control diet-induced bone loss might be beneficial in patients with preexisting bone inflammatory conditions.     

Deep-freeze preservation of cranial bones for future cranioplasty: nine years of experience in Soroka University Medical Center

Background Decompressive craniectomy is routinely performed in many neurosurgical centers to treat intracranial hypertension refractory to medical therapy as a result of head trauma, CVA or various brain tumors. When the patient survives his illness, cranioplasty with autologous bone graft or other reconstructive materials is considered to repair the skull defect. Objective This prospective study reviews the cases of decompressive craniectomies followed by later cranioplasty undertaken at our institute through the years 1996 and 2005 and describes the method used for preservation of removed bone flaps for future cranioplasty. Subjects and methods Sixty-eight patients underwent decompressive craniectomies since 1996. A protocol was designed to prepare the removed bone flaps for deep freeze preservation. After removal, the bone flaps were transferred to the skin bank at our institution within 6 h, gently rinsed using 1–3 liters of sterile saline (0.9% NaCl) supplemented with antibiotics (neomycin, 2 mM) with no dimethylsulfoxide (DMSO), then flaps were wrapped in two layers of sterile plastic coverage and preserved at −80°C. Results The patient’s population will be presented. Since 1996 we have performed 12 cranioplasties using deep-freeze preserved autologous bone graft. It took a rather long learning period, beginning with a single patient per year and continued with several others. Up to now, no case of infection, osteomyelitis or bone resorption following cranioplasty have occurred. Conclusion Deep-freeze preservation of autologous bone grafts to reconstruct skull defects after decompressive craniectomy is a useful procedure and has a low revision rate. N. Grossman: deceased 23 December 2006.     

Computed tomography in the diagnosis of soft tissue changes in patients with chronic osteomyelitis of the femur

Computed tomography was used to study soft tissues of the femur in 78 patients with its chronic recurrent osteomyelitis and the studied parameters were estimated. The fibrous changes were found to be more pronounced in posttraumatic osteomyelitis; the thickening of the subcutaneous fat and its atrophy were equally marked; the density gradients between the intact muscle, infiltrated muscle tissue, and purulent leakage were calculated.     

Osteomyelitis in a 265-million-year-old titanosuchid (Dinocephalia,Therapsida)

Abstract

Analytical palaeohistology techniques have allowed a better understanding of the microstructure of fossil bone, as well as of bone pathologies of extinct animals. Osteomyelitis is one of the oldest identified bone pathologies, occurring in Synapsida dating back as far as the Lower Permian. Here we show the presence of this pathology in the femur of Jonkeria parva, an omnivorous titanosuchid from the Tapinocephalus Assemblage Zone of the Karoo Basin of South Africa. The pathology is characterised by bony radial spicules growing perpendicular to the normal orientation of the unaffected fibrolamellar bone tissue, and shows localised increase in vascular canal size. Puncture marks on the femur suggests that an attack by a predator may have resulted in a bacterial infection that caused contiguous and subjacent osteomyelitis.     

Chronic arthritis leads to disturbances in the bone collagen network

Introduction

In this study we used a mice model of chronic arthritis to evaluate if bone fragility induced by chronic inflammation is associated with an imbalance in bone turnover and also a disorganization of the bone type I collagen network.

Methods

Serum, vertebrae and femur bones were collected from eight-month-old polyarthritis SKG mice and controls. Strength of the femoral bones was evaluated using three-point bending tests and density was assessed with a pycnometer. Bone turnover markers carboxy-terminal collagen cross-linking telopeptides (CTX-I) and amino-terminal propeptide of type I procollagen (PINP) were measured in serum. The organization and density of bone collagen were analyzed in vertebrae using second-harmonic generation (SHG) imaging with a two-photon microscope and trabecular bone microstructure was assessed by scanning electron microscope (SEM).

Results

Femoral bones of SKG mice revealed increased fragility expressed by deterioration of mechanical properties, namely altered stiffness (P = 0.007) and reduced strength (P = 0.006), when compared to controls. Accordingly, inter-trabecular distance and trabecular thickness as observed by SEM were reduced in SKG mice. PINP was significantly higher in arthritic mice (9.18 ± 3.21 ng/ml) when compared to controls (1.71 ± 0.53 ng/ml, P < 0.001). Bone resorption marker CTX-I was 9.67 ± 3.18 ng/ml in arthritic SKG mice compared to 6.23 ± 4.11 ng/ml in controls (P = 0.176). The forward-to-backward signal ratio measured by SHG was higher in SKG animals, reflecting disorganized matrix and loose collagen structure, compared to controls.

Conclusions

We have shown for the first time that chronic arthritis by itself impairs bone matrix architecture, probably due to disturbed bone remodeling and increased collagen turnover. This effect might predispose patients to bone fragility fractures.     

Allometry in the distribution of material properties and geometry of the felid skull: why larger species may need to change and how they may achieve it

Extant members of the cat family (Felidae) have been considered behaviourally and morphologically conservative, i.e., despite great differences in size, there is relatively little variation in either the shape of the felid skull and dentition across species, or in the way in which these structures are used to kill and dismember prey. Consequently felids have been considered an appropriate focus for a number of investigations into the influence of allometry on craniomandibular mechanics and morphology. However, although previous treatments have considered the role of shape, they have not investigated the influence of differences in the distribution of relatively stiff cortical and more compliant cancellous bone on performance. Here, using models that incorporate material properties for both cortical and cancellous bone, we apply three-dimensional (3D) finite element analysis (FEA) to models representing the skulls of seven extant felid species. Our objectives being to determine allometric trends regarding both overall geometry and the relative distributions of cortical and cancellous bone tissue. We also more comprehensively assess variation in the efficiency with which muscular force is converted to bite force and the capacity to resist associated stresses. Our results show that the cheetah (Acinonyx jubatus) may be exceptional regarding both the efficiency with which muscular force is converted to bite force and the distribution of stress. We found a negative allometric trend between cortical bone volume and total skull bone volume, and positive allometry between the total skull bone volume and skull surface area. Results gained from mathematical modelling of beam analogies suggest that these trends reflect a need for larger species to respond to physical challenges associated with increased size, and, that changes in skull shape, bone composition, or a combination of both may be required to accommodate these challenges. With geometrical scaling stress increases by the same factor, and displacement by the same factor squared, but the ultimate failure stress of the material is invariant. We find that as species become larger, overall skull bone volume relative to surface area increases by adding a higher proportion of less dense and more compliant cancellous bone. This results in an increased cross-sectional area and second moment of inertia, which acts to reduce the overall stresses. An overall saving in mass is a likely additional consequence. Although we do find evidence that skull stiffness does diminish with size, we also argue that this is at least in part mitigated through the influence of these allometric trends. We further suggest that these trends and the explanations for them may be universal for vertebrates.     

Staphylococcus epidermidis serine–aspartate repeat protein G (SdrG) binds to osteoblast integrin alpha V beta 3

Staphylococcus epidermidis is the leading etiologic agent of orthopaedic implant infection. Contamination of the implanted device during insertion allows bacteria gain entry into the sterile bone environment leading to condition known as osteomyelitis. Osteomyelitis is characterised by weakened bones associated with progressive bone loss. The mechanism through which S. epidermidis interacts with bone cells to cause osteomyelitis is poorly understood. We demonstrate here that S. epidermidis can bind to osteoblasts in the absence of matrix proteins. S. epidermidis strains lacking the cell wall protein SdrG had a significantly reduced ability to bind to osteoblasts. Consistent with this, expression of SdrG in Lactococcus lactis resulted in significantly increased binding to the osteoblasts. Protein analysis identified that SdrG contains a potential integrin recognition motif. αVβ3 is a major integrin expressed on osteoblasts and typically recognises RGD motifs in its ligands. Our results demonstrate that S. epidermidis binds to recombinant purified αVβ3, and that a mutant lacking SdrG failed to bind. Blocking αVβ3 on osteoblasts significantly reduced binding to S. epidermidis. These studies are the first to identify a mechanism through which S. epidermidis binds to osteoblasts and potentially offers a mechanism through which implant infection caused by S. epidermidis leads to osteomyelitis.     

Interspecific Variation in Compensatory Regrowth to Herbivory Associated with Soil Nutrients in Three Ficus (Moraceae) Saplings

Plant compensatory regrowth is an induced process that enhances plant tolerance to herbivory. Plant behavior against herbivores differs between species and depends on resource availability, thus making general predictions related to plant compensatory regrowth difficult. To understand how soil nutrients determine the degree of compensatory regrowth for different plant species, we selected saplings of three Ficus species and treated with herbivore insects and artificial injury in both glasshouse conditions and in the field at two soil nutrient levels. Compensatory regrowth was calculated by biomass, relative growth rate and photosynthetic characteristics. A similar pattern was found in both the glasshouse and in the field for species F. hispida, where overcompensatory regrowth was triggered only under fertile conditions, and full compensatory regrowth occurred under infertile conditions. For F. auriculata, overcompensatory regrowth was stimulated only under infertile conditions and full compensatory regrowth occurred under fertile conditions. Ficus racemosa displayed full compensatory regrowth in both soil nutrient levels, but without overcompensatory regrowth following any of the treatments. The three Ficus species differed in biomass allocation following herbivore damage and artificial injury. The root/shoot ratio of F. hispida decreased largely following herbivore damage and artificial injury, while the root/shoot ratio for F. auriculata increased against damage treatments. The increase of shoot and root size for F. hispida and F. auriculata, respectively, appeared to be caused by a significant increase in photosynthesis. The results indicated that shifts in biomass allocation and increased photosynthesis are two of the mechanisms underlying compensatory regrowth. Contrasting patterns among the three Ficus species suggest that further theoretical and empirical work is necessary to better understand the complexity of the plant responses to herbivore damage.