Pamela J. VandeVord

Improved tissue-engineered bone regeneration by endothelial cell mediated vascularization

Natural bone growth greatly depends on the precedent vascular network that supplies oxygen and essential nutrients and removes metabolites. Likewise, it is crucial for tissue-engineered bone to establish a vascular network that temporally precedes new bone formation, and spatially originates from within the graft. In order to recapitulate physiological skeletal development, we have developed a complex bone graft to repair rat bone defects. We have demonstrated that endothelial cells and osteoblasts (identified by cell morphology, quantification of specific marker antigens, calcium deposition and capillary-like growth) were able to differentiate and expand from donor rat bone marrow mononuclear cell populations. The biocompatibilities of poly-epsilon-caprolactone (PCL)-hydroxyapatite (HA) composites used for graft fabrication were evaluated at different component ratios to identify the optimal and support of cellular viability and functions for endothelial cells and osteoblasts. Using point-injection and low-pressure techniques, seeded endothelial cells and osteoblasts were able to assemble into microvascular networks and form bony matrix in grafts. The exogenous origination of these cells and their contribution to the vascularization and osteogenesis was confirmed using sex-mismatch implantation and Y chromosome tracking. By pre-seeding with endothelial cells, the resulting vascularization was able to promote osteogenesis, prevent ischemic necrosis and improve the mechanical properties in engineered bone tissue. Taken together, the results indicated that the integration of complex cell populations with composite scaffold materials provided an effective technique to improve osteogenesis in engineered bone graft. These findings suggest that hybrid grafts have great potential for clinical use to treat large bone defects.

Intracranial pressure increases during exposure to a shock wave

Traumatic brain injuries (TBI) caused by improvised explosive devices (IEDs) affect a significant percentage of surviving soldiers wounded in Iraq and Afghanistan. The extent of a blast TBI, especially initially, is difficult to diagnose, as internal injuries are frequently unrecognized and therefore underestimated, yet problems develop over time. Therefore it is paramount to resolve the physical mechanisms by which critical stresses are inflicted on brain tissue from blast wave encounters with the head. This study recorded direct pressure within the brains of male Sprague-Dawley rats during exposure to blast. The goal was to understand pressure wave dynamics through the brain. In addition, we optimized in vivo methods to ensure accurate measurement of intracranial pressure (ICP). Our results demonstrate that proper sealing techniques lead to a significant increase in ICP values, compared to the outside overpressure generated by the blast. Further, the values seem to have a direct relation to a rats size and age: heavier, older rats had the highest ICP readings. These findings suggest that a global flexure of the skull by the transient shockwave is an important mechanism of pressure transmission inside the brain.

Promotion of osteogenesis in tissue-engineered bone by pre-seeding endothelial progenitor cells-derived endothelial cells

In addition to a biocompatible scaffold and an osteogenic cell population, tissue‐engineered bone requires an appropriate vascular bed to overcome the obstacle of nutrient and oxygen transport in the 3D structure. We hypothesized that the addition of endothelial cells (ECs) may improve osteogenesis and prevent necrosis of engineered bone via effective neovascularization. Osteoblasts and ECs were differentiated from bone marrow of BALB/c mice, and their phenotypes were confirmed prior to implantation. Cylindrical porous polycaprolactone (PCL)‐hydroxyapatite (HA) scaffolds were synthesized. ECs were seeded on scaffolds followed by seeding of osteoblasts in the EC‐OB group. In the OB group, scaffolds were only seeded with osteoblasts. The cell‐free scaffolds were denoted as control group. A 0.4‐cm‐long segmental femur defect was established and replaced with the grafts. The grafts were evaluated histologically at 6 weeks postimplantation. In comparison with the OB group, the EC‐OB group resulted in a widely distributed capillary network, osteoid generated by osteoblasts and absent ischemic necroses. Pre‐seeding scaffold with ECs effectively promoted neovascularization in grafts, prevented the ischemic necrosis, and improved osteogenesis. The integration of bone marrow‐derived ECs and osteoblasts in porous scaffold is a useful strategy to achieve engineered bone.

Skull Flexure as a Contributing Factor in the Mechanism of Injury in the Rat when Exposed to a Shock Wave

The manner in which energy from an explosion is transmitted into the brain is currently a highly debated topic within the blast injury community. This study was conducted to investigate the injury biomechanics causing blast-related neurotrauma in the rat. Biomechanical responses of the rat head under shock wave loading were measured using strain gauges on the skull surface and a fiber optic pressure sensor placed within the cortex. MicroCT imaging techniques were applied to quantify skull bone thickness. The strain gauge results indicated that the response of the rat skull is dependent on the intensity of the incident shock wave; greater intensity shock waves cause greater deflections of the skull. The intracranial pressure (ICP) sensors indicated that the peak pressure developed within the brain was greater than the peak side-on external pressure and correlated with surface strain. The bone plates between the lambda, bregma, and midline sutures are probable regions for the greatest flexure to occur. The data provides evidence that skull flexure is a likely candidate for the development of ICP gradients within the rat brain. This dependency of transmitted stress on particular skull dynamics for a given species should be considered by those investigating blast-related neurotrauma using animal models.

Blast induces oxidative stress, inflammation, neuronal loss and subsequent short-term memory impairment in rats.

Molecular and cellular mechanisms of brain injury after exposure to blast overpressure (BOP) are not clearly known. The present study hypothesizes that pro-oxidative and pro-inflammatory pathways in the brain may be responsible for neuronal loss and behavioral deficits following BOP exposure. Male Sprague-Dawley rats were anesthetized and exposed to calibrated BOP of 129.23±3.01kPa while controls received only anesthesia. In situ dihydroethidium fluorescence staining revealed that BOP significantly increased the production of reactive oxygen species in the brain. In addition, real-time reverse transcriptase-polymerase chain reaction, immunofluorescence staining and enzyme-linked immunosorbent assay demonstrated a significant up-regulation of mRNA and protein expressions of pro-inflammatory mediators, such as interferon-γ and monocyte chemoattractant protein-1, in brains collected from BOP-exposed animals compared with the controls. Furthermore, immunoreactivity of neuronal nuclei in brains indicated that fewer neurons were present following BOP exposure. Moreover, novel object recognition paradigm showed a significant impairment in the short-term memory at 2weeks following BOP exposure. These results suggest that pro-oxidative and pro-inflammatory environments in the brain could play a potential role in BOP-induced neuronal loss and behavioral deficits. It may provide a foundation for defining a molecular and cellular basis of the pathophysiology of blast-induced neurotrauma (BINT). It will also contribute to the development of new therapeutic approaches selectively targeting these pathways, which have great potential in the diagnosis and therapy of BINT.

Antinuclear antibodies as potential markers of lung cancer

There are multiple case reports of antinuclear antibodies (ANAs) in patients with malignancies, yet to date there has not been a systematic survey of ANAs in lung cancer. We have previously reported that autoantibodies to collagen antigens resembling those found in the connective tissue diseases are consistently detected in the sera from lung cancer patients. In this work, we looked for the presence of ANAs in the sera from these same patients. Sera from 64 patients with lung cancer and 64 subjects without a history of cancer were retrospectively tested for reactivity on immunoblots of nuclear extracts of HeLa, small cell carcinoma, squamous cell carcinoma, adenocarcinoma, large cell carcinoma of the lung, and of normal lung cells. Associations were sought between the reactivities on immunoblots and lung cancer cell type, diagnosis, and progression-free survival by the method of classification and regression trees (CARTs). Cross-validated CART analyses indicated that reactivities to certain bands in immunoblots are associated with different types of lung cancer. Some of these autoantibodies were associated with a prolonged survival without disease progression. Our data suggest that autoimmunity is often a prominent feature of lung cancer and that molecular characterization of these antigens may lead to the discovery of proteins with diagnostic and prognostic value.

Blast-induced tinnitus and hearing loss in rats: behavioral and imaging assays.

Abstract The current study used a rat model to investigate the underlying mechanisms of blast-induced tinnitus, hearing loss, and associated traumatic brain injury (TBI). Seven rats were used to evaluate behavioral evidence of tinnitus and hearing loss, and TBI using magnetic resonance imaging following a single 10-msec blast at 14 psi or 194 dB sound pressure level (SPL). The results demonstrated that the blast exposure induced early onset of tinnitus and central hearing impairment at a broad frequency range. The induced tinnitus and central hearing impairment tended to shift towards high frequencies over time. Hearing threshold measured with auditory brainstem responses also showed an immediate elevation followed by recovery on day 14, coinciding with behaviorally-measured results. Diffusion tensor magnetic resonance imaging results demonstrated significant damage and compensatory plastic changes to certain auditory brain regions, with the majority of changes occurring in the inferior colliculus and medial geniculate body. No significant microstructural changes found in the corpus callosum indicates that the currently adopted blast exposure mainly exerts effects through the auditory pathways rather than through direct impact onto the brain parenchyma. The results showed that this animal model is appropriate for investigation of the mechanisms underlying blast-induced tinnitus, hearing loss, and related TBI. Continued investigation along these lines will help identify pathology with injury/recovery patterns, aiding development of effective treatment strategies.

Blast-induced neurotrauma leads to neurochemical changes and neuronal degeneration in the rat hippocampus.

Blast‐induced neurotrauma is a major concern because of the complex expression of neuropsychiatric disorders after exposure. Disruptions in neuronal function, proximal in time to blast exposure, may eventually contribute to the late emergence of clinical deficits. Using magic angle spinning 1H MRS and a rodent model of blast‐induced neurotrauma, we found acute (24–48 h) decreases in succinate, glutathione, glutamate, phosphorylethanolamine and γ‐aminobutyric acid, no change in N‐acetylaspartate and increased glycerophosphorylcholine, alterations consistent with mitochondrial distress, altered neurochemical transmission and increased membrane turnover. Increased levels of the apoptotic markers Bax and caspase‐3 suggested active cell death, consistent with increased FluoroJade B staining in the hippocampus. Elevated levels of glial fibrillary acidic protein suggested ongoing inflammation without diffuse axonal injury measured by no change in β‐amyloid precursor protein. In conclusion, blast‐induced neurotrauma induces a metabolic cascade associated with neuronal loss in the hippocampus in the acute period following exposure. Copyright

GDNF blended chitosan nerve guides: an in vivo study.

Chitosan nerve guides are currently being utilized to repair damaged or injured peripheral nerves. To enhance the nerve regeneration process, instead of using the material alone, researchers are focusing on blending different proteins or molecules with chitosan to facilitate nerve repair and regeneration. In our study, we have blended chitosan with glial cell-line derived neurotrophic factor and laminin within our nerve guides (GLC). The rat sciatic nerve injury model was used to test these nerve guides and histologically evaluated at 6, 9, and 12 weeks. Histologically at 6 weeks, the axon area and myelination are significantly higher in the GLC group compared with the controls. However, at 9 and 12 weeks control groups matched the GLC values. Thus the histological results indicate that GLC nerve guides can enhance the nerve regeneration process during the initial stages of nerve repair.

Nanopatterning effects on astrocyte reactivity.

An array of design strategies have been targeted toward minimizing failure of implanted microelectrodes by minimizing the chronic glial scar around the microelectrode under chronic conditions. Current approaches toward inhibiting the initiation of glial scarring range from altering the geometry, roughness, size, shape, and materials of the device. Studies have shown materials which mimic the nanotopography of the natural environment in vivo will consequently result in an improved biocompatible response. Nanofabrication of electrode arrays is being pursued in the field of neuronal electrophysiology to increase sampling capabilities. Literature shows a gap in research of nanotopography influence in the reduction of astrogliosis. The aim of this study was to determine optimal feature sizes for neural electrode fabrication, which was defined as eliciting a nonreactive astrocytic response. Nanopatterned surfaces were fabricated with nanoimprint lithography on poly(methyl methacrylate) surfaces. The rate of protein adsorption, quantity of protein adsorption, cell alignment, morphology, adhesion, proliferation, viability, and gene expression was compared between nanopatterned surfaces of different dimensions and non-nanopatterned control surfaces. Results of this study revealed that 3600 nanopatterned surfaces elicited less of a response when compared with the other patterned and non-nanopatterned surfaces. The surface instigated cell alignment along the nanopattern, less protein adsorption, less cell adhesion, proliferation and viability, inhibition of glial fibrillary acidic protein, and mitogen-activated protein kinase kinase 1 compared with all other substrates tested.