Streptococcus agalactiae Infects Glial Cells and Invades the Central Nervous System via the Olfactory and Trigeminal Nerves.

Streptococcus agalactiae causes neonatal meningitis and can also infect the adult central nervous system (CNS). S. agalactiae can cross the blood-brain barrier but may also reach the CNS via other paths. Several species of bacteria can directly invade the CNS via the olfactory and trigeminal nerves, which extend between the nasal cavity and brain and injury to the nasal epithelium can increase the risk/severity of infection. Preterm birth is associated with increased risk of S. agalactiae infection and with nasogastric tube feeding. The tubes, also used in adults, can cause nasal injuries and may be contaminated with bacteria, including S. agalactiae. We here investigated whether S. agalactiae could invade the CNS after intranasal inoculation in mice. S. agalactiae rapidly infected the olfactory nerve and brain. Methimazole-mediated model of nasal epithelial injury led to increased bacterial load in these tissues, as well as trigeminal nerve infection. S. agalactiae infected and survived intracellularly in cultured olfactory/trigeminal nerve- and brain-derived glia, resulting in cytokine production, with some differences between glial types. Furthermore, a non-capsulated S. agalactiae was used to understand the role of capsule on glial cells interaction. Interestingly, we found that the S. agalactiae capsule significantly altered cytokine and chemokine responses and affected intracellular survival in trigeminal glia. In summary, this study shows that S. agalactiae can infect the CNS via the nose-to-brain path with increased load after epithelial injury, and that the bacteria can survive in glia.

Chlamydia pneumoniae can infect the central nervous system via the olfactory and trigeminal nerves and contributes to Alzheimer's disease risk.

Chlamydia pneumoniae is a respiratory tract pathogen but can also infect the central nervous system (CNS). Recently, the link between C. pneumoniae CNS infection and late-onset dementia has become increasingly evident. In mice, CNS infection has been shown to occur weeks to months after intranasal inoculation. By isolating live C. pneumoniae from tissues and using immunohistochemistry, we show that C. pneumoniae can infect the olfactory and trigeminal nerves, olfactory bulb and brain within 72 h in mice. C. pneumoniae infection also resulted in dysregulation of key pathways involved in Alzheimer's disease pathogenesis at 7 and 28 days after inoculation. Interestingly, amyloid beta accumulations were also detected adjacent to the C. pneumoniae inclusions in the olfactory system. Furthermore, injury to the nasal epithelium resulted in increased peripheral nerve and olfactory bulb infection, but did not alter general CNS infection. In vitro, C. pneumoniae was able to infect peripheral nerve and CNS glia. In summary, the nerves extending between the nasal cavity and the brain constitute invasion paths by which C. pneumoniae can rapidly invade the CNS likely by surviving in glia and leading to Aß deposition.

Induction of Complete Transection-Type Spinal Cord Injury in Mice.

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, such as cell transplantation methods, are being researched to overcome the debilitating disabilities arising from SCI. Most pre-clinical animal trials are conducted in rodent models of SCI. While rat models of SCI have been widely used, mouse models have received less attention, even though mouse models can have significant advantages over rat models. The small size of mice equates to lower animal maintenance costs than for rats, and the availability of numerous transgenic mouse models is advantageous for many types of studies. Inducing repeatable and precise injury in the animals is the primary challenge for SCI research, which in small rodents requires high-precision surgery. The transection-type injury model has been a commonly used injury model over the last decade for transplantation-based therapeutic research, however a standardized method for inducing a complete transection-type injury in mice does not exist. We have developed a surgical protocol for inducing a complete transection type injury in C57BL/6 mice at thoracic vertebral level 10 (T10). The procedure uses a small tip drill instead of rongeurs to precisely remove the lamina, after which a thin blade with rounded cutting edge is used to induce the spinal cord transection. This method leads to reproducible transection-type injury in small rodents with minimal collateral muscle and bone damage and therefore minimizes confounding factors, specifically where behavioral functional outcomes are analyzed.

Widespread differential maternal and paternal genome effects on fetal bone phenotype at mid-gestation.

Parent-of-origin-dependent (epi)genetic factors are important determinants of prenatal development that program adult phenotype. However, data on magnitude and specificity of maternal and paternal genome effects on fetal bone are lacking. We used an outbred bovine model to dissect and quantify effects of parental genomes, fetal sex, and nongenetic maternal effects on the fetal skeleton and analyzed phenotypic and molecular relationships between fetal muscle and bone. Analysis of 51 bone morphometric and weight parameters from 72 fetuses recovered at day 153 gestation (54% term) identified six principal components (PC1-6) that explained 80% of the variation in skeletal parameters. Parental genomes accounted for most of the variation in bone wet weight (PC1, 72.1%), limb ossification (PC2, 99.8%), flat bone size (PC4, 99.7%), and axial skeletal growth (PC5, 96.9%). Limb length showed lesser effects of parental genomes (PC3, 40.8%) and a significant nongenetic maternal effect (gestational weight gain, 29%). Fetal sex affected bone wet weight (PC1, p < 0.0001) and limb length (PC3, p < 0.05). Partitioning of variation explained by parental genomes revealed strong maternal genome effects on bone wet weight (74.1%, p < 0.0001) and axial skeletal growth (93.5%, p < 0.001), whereas paternal genome controlled limb ossification (95.1%, p < 0.0001). Histomorphometric data revealed strong maternal genome effects on growth plate height (98.6%, p < 0.0001) and trabecular thickness (85.5%, p < 0.0001) in distal femur. Parental genome effects on fetal bone were mirrored by maternal genome effects on fetal serum 25-hydroxyvitamin D (96.9%, p < 0.001) and paternal genome effects on alkaline phosphatase (90.0%, p < 0.001) and their correlations with maternally controlled bone wet weight and paternally controlled limb ossification, respectively. Bone wet weight and flat bone size correlated positively with muscle weight (r = 0.84 and 0.77, p < 0.0001) and negatively with muscle H19 expression (r = -0.34 and -0.31, p < 0.01). Because imprinted maternally expressed H19 regulates growth factors by miRNA interference, this suggests muscle-bone interaction via epigenetic factors.

Maternal and paternal genomes differentially affect myofibre characteristics and muscle weights of bovine fetuses at midgestation.

Postnatal myofibre characteristics and muscle mass are largely determined during fetal development and may be significantly affected by epigenetic parent-of-origin effects. However, data on such effects in prenatal muscle development that could help understand unexplained variation in postnatal muscle traits are lacking. In a bovine model we studied effects of distinct maternal and paternal genomes, fetal sex, and non-genetic maternal effects on fetal myofibre characteristics and muscle mass. Data from 73 fetuses (Day153, 54% term) of four genetic groups with purebred and reciprocal cross Angus and Brahman genetics were analyzed using general linear models. Parental genomes explained the greatest proportion of variation in myofibre size of Musculus semitendinosus (80-96%) and in absolute and relative weights of M. supraspinatus, M. longissimus dorsi, M. quadriceps femoris and M. semimembranosus (82-89% and 56-93%, respectively). Paternal genome in interaction with maternal genome (P<0.05) explained most genetic variation in cross sectional area (CSA) of fast myotubes (68%), while maternal genome alone explained most genetic variation in CSA of fast myofibres (93%, P<0.01). Furthermore, maternal genome independently (M. semimembranosus, 88%, P<0.0001) or in combination (M. supraspinatus, 82%; M. longissimus dorsi, 93%; M. quadriceps femoris, 86%) with nested maternal weight effect (5-6%, P<0.05), was the predominant source of variation for absolute muscle weights. Effects of paternal genome on muscle mass decreased from thoracic to pelvic limb and accounted for all (M. supraspinatus, 97%, P<0.0001) or most (M. longissimus dorsi, 69%, P<0.0001; M. quadriceps femoris, 54%, P<0.001) genetic variation in relative weights. An interaction between maternal and paternal genomes (P<0.01) and effects of maternal weight (P<0.05) on expression of H19, a master regulator of an imprinted gene network, and negative correlations between H19 expression and fetal muscle mass (P<0.001), suggested imprinted genes and miRNA interference as mechanisms for differential effects of maternal and paternal genomes on fetal muscle.

Cages augmented with mineralized collagen and platelet-rich plasma as an osteoconductive/inductive combination for interbody fusion.

STUDY DESIGN: After anterior cervical discectomy, fusion was radiologically, biomechanically, and histologically assessed in a sheep spine fusion model. OBJECTIVE: To evaluate the efficacy of a platelet-rich plasma (PRP) application combined with a mineralized collagen matrix (MCM) as an alternative to autologous cancellous iliac crest bone grafts in a spine fusion model. SUMMARY OF BACKGROUND DATA: PRP has the ability to stimulate bone and tissue healing. MCM is a recently developed osteoconductive material. Up to now, no comparative evaluation of PRP in combination with a MCM at the cervical spine has been performed in vivo. METHODS: Twenty-four sheep (N = 8/group) underwent C3/4 discectomy and fusion: group 1, titanium cage filled with autologous cancellous iliac crest bone graft; group 2, titanium cage filled with MCM; and group 3, titanium cage filled with MCM and PRP. Radiographic evaluation was performed before surgery and after 1, 2, 4, 8, and 12 weeks, respectively. After 12 weeks, fusion sites were evaluated using functional radiographic views and quantitative computed tomographic scans to assess bone mineral density. Furthermore, histomorphologic and histomorphometrical analyses were performed to evaluate fusion. RESULTS: In comparison with the titanium cage group filled with autologous cancellous iliac crest bone grafts representing the control group, MCM-alone group showed a slightly lower fusion rate in the radiographic and the histomorphometrical analysis. The addition of PRP could not enhance this finding. There was no significant difference between MCM and MCM + PRP group in radiologic and histologic findings. CONCLUSION: The MCM alone is not able to replace autologous bone grafts. Early activation of the platelets by calcium, which is released from mineralized collagen, could be the reason for the insufficient osteoinductive effect of PRP. In consequence, the combined application of mineralized collagen and PRP had no significant osteoinductive effect in this model.

Sphene ceramics for orthopedic coating applications: an in vitro and in vivo study.

The host response to titanium alloy (Ti-6Al-4V) is not always favorable as a fibrous layer may form at the skeletal tissue-device interface, causing aseptic loosening. Recently, sphene (CaTiSiO(5)) ceramics were developed by incorporating Ti in the Ca-Si system, and found to exhibit improved chemical stability. The aim of this study is to evaluate the in vitro response of human osteoblast-like cells, human osteoclasts and human microvascular endothelial cells to sphene ceramics and determine whether coating Ti-6Al-4V implants with sphene enhances anchorage to surrounding bone. The study showed that sphene ceramics support human osteoblast-like cell attachment with organized cytoskeleton structure and express increased mRNA levels of osteoblast-related genes. Sphene ceramics were able to induce the differentiation of monocytes to form functional osteoclasts with the characteristic features of f-actin and alpha(v)beta(3) integrin, and express osteoclast-related genes. Human endothelial cells were also able to attach and express the endothelial cell markers ZO-1 and VE-Cadherin when cultured on sphene ceramics. Histological staining, enzyme histochemistry and immunolabelling were used for identification of mineralized bone and bone remodelling around the coated implants. Ti-6Al-4V implants coated with sphene showed new bone formation and filled the gap between the implants and existing bone in a manner comparable to that of the hydroxyapatite coatings used as control. The new bone was in direct contact with the implants, whereas fibrous tissue formed between the bone and implant with uncoated Ti-6Al-4V. The in vivo assessment of sphene-coated implants supports our in vitro observation and suggests that they have the ability to recruit osteogenic cells, and thus support bone formation around the implants and enhance osseointegration.

Biomechanical testing of the lumbar facet interference screw.

STUDY DESIGN: An in vitro study was conducted to determine the biomechanical properties of a new simple, percutaneous, posterior fixation technique for the lumbar spine involving a new implant, the so-called Lumbar Facet Interference Screw. OBJECTIVES: The purpose of this study was to compare the biomechanical properties of this new fixation device with translaminar and pedicle screw fixation. SUMMARY OF BACKGROUND DATA: Several techniques were described to perform a minimal invasive posterior stabilization of the lumbar spine after an anterior lumbar interbody fusion procedure. Yet, due to the high complexity of these minimally invasive surgical procedures, currently, hardly any of these percutaneous posterior fixation techniques is carried out routinely. METHODS: Ten human lumbar spines were tested in flexion, extension, axial rotation, and lateral bending using a nonconstrained testing method. First, all motion segments were evaluated intact (group 1). After complete discectomy of L4-L5, the following stabilization techniques were tested sequentially (n = 10/group): group 2: "stand-alone" cage; group 3: cage plus translaminar screws; group 4: cage plus Lumbar Facet Interference Screw; and group 5: cage plus pedicle screws. Stiffness, ranges of motion, and neutral and elastic zones were determined. RESULTS: In comparison to the intact motion segment, the "stand-alone" cage showed a significantly higher (P < 0.05) range of motion, neutral zone, and elastic zone and a significantly lower (P < 0.05) stiffness in extension and rotation. Generally, all fixation techniques using cages plus posterior stabilization decreased range of motion, neutral zone, and elastic zone and increased stiffness in comparison to the "stand-alone" cage group. There was no significant difference between the cage plus interference screw and the cage plus translaminar screw group in all test modes. In comparison to the 2 facet joint stabilization techniques, pedicle screw stabilization decreased (P < 0.01) range of motion, neutral zone, and elastic zone and increased (P < 0.01) stiffness significantly in flexion and rotation. CONCLUSIONS: Results of this study indicate that the new Lumbar Facet Interference Screw fixation yields initial biomechanical stability similar to translaminar screw fixation, yet inferior biomechanical stability compared to pedicle screw fixation. Although these results are encouraging, additional biomechanical studies including cyclic loading tests have to evaluate the mid- and long-term stabilization capacity of this new minimally invasive fixation technique before human application.

Bioabsorbable interbody cages in a sheep cervical spine fusion model.

STUDY DESIGN: An experimental study using a sheep cervical spine interbody fusion model. OBJECTIVES: To compare interbody fusion of an autologous tricortical iliac crest bone graft with two bioabsorbable cages and to determine whether there are differences between the three interbody fusion techniques in 1) the ability to preserve postoperative distraction, 2) the biomechanical stability, and 3) the histologic characteristics of intervertebral bone matrix formation. SUMMARY AND BACKGROUND DATA: Bioabsorbable cages would be beneficial compared with metallic cages; however, currently no suitable bioabsorbable interbody fusion cage is available. METHOD: Twenty-four sheep underwent C3/C4 discectomy and fusion. The following stabilization techniques were used: Group 1) autologous tricortical iliac crest bone graft (n = 8); Group 2) bioabsorbable cage made of 70/30 poly(l-lactide-co-d,l-lactide) (experimental) filled with autologous cancellous bone graft (n = 8); Group 3) bioabsorbable cage made of a polymer-calciumphosphate composite (Biomet Europe, Dordrecht, The Netherlands) filled with autologous cancellous bone graft (n = 8). Radiographic scans to determine disc space height were performed before and after surgery and after 1, 2, 4, 8, and 12 weeks, respectively. After 12 weeks, animals were killed, and fusion sites were evaluated using functional radiographic views in flexion and extension. Quantitative computed tomographic scans were used to assess bone mineral density, bone mineral content, and bony callus volume. Biomechanical testing was performed in flexion, extension, axial rotation, and lateral bending to determine stiffness, ROM, and neutral and elastic zone. Histomorphological and histomorphometrical analysis were performed to evaluate fusion and foreign body reactions associated with the bioabsorbable cages. RESULTS: Over a 12-week period, the polymer-calciumphosphate composite cage showed significantly higher values for disc space height compared with the bone graft and the poly(l-lactide-co-d,l-lactide) cage. Additionally, the polymer-calciumphosphate composite cage demonstrated a significantly higher stiffness and lower ROM, neutral zone, and elastic zone in axial rotation and lateral bending than any other group. However, quantitative computed tomographic scans demonstrated cracks in six of the eight polymer-calciumphosphate composite cages after 12 weeks. Histologically, the highest bone volume/total volume ratio and the highest fusion rate were found in the polymer-calciumphosphate composite cage group. Although the poly(l-lactide-co-d,l-lactide) cage showed grade I through III foreign body reactions in all fusion areas, only two animals developed grade I foreign body reactions with the polymer-calciumphosphate composite cage. CONCLUSION: After 12 weeks, there was no significant difference between the bioabsorbable poly(l-lactide-co-d,l-lactide) cage and the tricortical bone graft. In comparison to the tricortical bone graft, the bioabsorbable polymer-calciumphosphate composite cage showed significantly better distractive properties, a significantly higher biomechanical stiffness, and an advanced interbody fusion; however, six of eight polymer-calciumphosphate composite cages cracked. Although the fate of the foreign body reactions and the cracks is currently unclear for both bioabsorbable cages, the early appearance of large osteolysis associated with use of the poly(l-lactide-co-d,l-lactide) cage allows skepticism regarding the value of this bioabsorbable implant.

Biomechanical comparison of bioabsorbable cervical spine interbody fusion cages.

STUDY DESIGN: In vitro biomechanical study of bioabsorbable cervical spine interbody fusion cages using a sheep model. OBJECTIVES: The purpose of this study was to evaluate the segmental stability provided by 2 new developed bioabsorbable cervical spine interbody fusion cages and to compare it with a tricortical iliac crest bone graft and a titanium meshed interbody fusion cage. Further, the biomechanical effect of an additional anterior plate instrumentation was determined. SUMMARY AND BACKGROUND DATA: Despite the initial favorable results, the long-term effects of metallic cage devices on spinal motion segments are still unknown. Furthermore, shortcomings of metallic cages like migration, adjacent level degeneration, stenotic myelopathy, and artifacts in postoperative radiologic assessment have already been reported. Bioabsorbable cages have been designed to avoid these complications. Currently, no information is available about the biomechanical properties of bioabsorbable cervical spine interbody fusion cages. METHODS: Forty sheep cervical spines (C2-C5) were tested in flexion, extension, axial rotation, and lateral bending with a nondestructive stiffness method using a nonconstrained testing apparatus. First, the motion segment C3-C4 was tested intact. After complete discectomy, the following groups were evaluated: autologous iliac crest bone graft, titanium mesh cylinder (Harms, DePuy AcroMed), bioabsorbable PDLLA-cage (experimental), and bioabsorbable Resorbon cage (Biomet Merck). Further, all implants were tested with an additional anterior plate instrumentation. The mean apparent stiffness, range of motion, neutral zone, and elastic zone were calculated from the corresponding load-displacement curves. RESULTS: No significant difference in range of motion and segmental stiffness among the tricortical iliac crest bone graft, meshed titanium Harms cage, and PDLLA-cage could be determined. The Resorbon cage significantly (P < 0.05) decreased range of motion and increased stiffness in rotation and flexion in comparisonto all tested implants and the intact motion segment. An additional anterior plate significantly (P < 0.05) decreased range of motion and increased stiffness in flexion and extension. CONCLUSION: In this study, bioabsorbable cages demonstrated biomechanical in vitro properties equal or superior to metallic cages. From the biomechanical point of view, bioabsorbable cages, especially the Resorbon cage, may be a viable alternative to current metallic interbody cage devices. However, animal experimental in vivo evaluation of bioabsorbable cervical spine interbody fusion cages still has to be performed.