Julie M. Hasenwinkel

Mechanical Characterization of the Injured Spinal Cord after Lateral Spinal Hemisection Injury in the Rat

The glial scar formed at the site of traumatic spinal cord injury (SCI) has been classically hypothesized to be a potent physical and biochemical barrier to nerve regeneration. One longstanding hypothesis is that the scar acts as a physical barrier due to its increased stiffness in comparison to uninjured spinal cord tissue. However, the information regarding the mechanical properties of the glial scar in the current literature is mostly anecdotal and not well quantified. We monitored the mechanical relaxation behavior of injured rat spinal cord tissue at the site of mid-thoracic spinal hemisection 2 weeks and 8 weeks post-injury using a microindentation test method. Elastic moduli were calculated and a modified standard linear model (mSLM) was fit to the data to estimate the relaxation time constant and viscosity. The SLM was modified to account for a spectrum of relaxation times, a phenomenon common to biological tissues, by incorporating a stretched exponential term. Injured tissue exhibited significantly lower stiffness and elastic modulus in comparison to uninjured control tissue, and the results from the model parameters indicated that the relaxation time constant and viscosity of injured tissue were significantly higher than controls. This study presents direct micromechanical measurements of injured spinal cord tissue post-injury. The results of this study show that the injured spinal tissue displays complex viscoelastic behavior, likely indicating changes in tissue permeability and diffusivity.

Raman spectroscopic investigation of spinal cord injury in a rat model

Raman spectroscopy was used to study temporal molecular changes associated with spinal cord injury (SCI) in a rat model. Raman spectra of saline-perfused, injured, and healthy rat spinal cords were obtained and compared. Two injury models, a lateral hemisection and a moderate contusion were investigated. The net fluorescence and the Raman spectra showed clear differences between the injured and healthy spinal cords. Based on extensive histological and biochemical characterization of SCI available in the literature, these differences were hypothesized to be due to cell death, demyelination, and changes in the extracellular matrix composition, such as increased expression of proteoglycans and hyaluronic acid, at the site of injury where the glial scar forms. Further, analysis of difference spectra indicated the presence of carbonyl containing compounds, hypothesized to be products of lipid peroxidation and acid catalyzed hydrolysis of glycosaminoglycan moieties. These results compared well with in vitro experiments conducted on chondroitin sulfate sugars. Since the glial scar is thought to be a potent biochemical barrier to nerve regeneration, this observation suggests the possibility of using near infrared Raman spectroscopy to study injury progression and explore potential treatments ex vivo, and ultimately monitor potential remedial treatments within the spinal cord in vivo.

A versatile mesoindentation system to evaluate the micromechanical properties of soft, hydrated substrates on a cellular scale.

It has become increasingly important to study mechanical properties of substrates on the cellular scale since cells sense and respond to changes in the microenvironment in which they are grown. To study the effects of mechanical substrate properties on the cellular scale, an existing microindentation system has been modified to perform indentation tests on highly hydrated polymeric substrates and tissues. The highly sensitive, modified indentation system, labeled as a mesoindenter, is versatile and can be customized to perform a variety of tests useful for studying tissue mechanics, stress relaxation in polymers, and interfacial adhesion phenomena. To validate the efficacy and accuracy of the system, soft, hydrated hydrogels made from agarose (1-5 wt %), poly(2-hydroxyethyl methacrylate) (p(HEMA)) (60-90% water), and unfixed, saline-perfused rat spinal cord tissue were tested. The results demonstrate that moduli vary with water content and are in line with previously published studies. We also demonstrate that the modulus of hydrogels is sensitive to the preload applied, with modulus increasing with preload. Stress relaxation indentation testing of p(HEMA) showed relaxation behavior that can be modeled using a heredity integral and standard linear model. The mesoindenter is versatile, capable of scanning and testing immersed samples, and easily customized to ascertain mechanical properties of substrates ranging from the kPa to GPa range.

Two-solution bone cements with cross-linked micro and nano-particles for vertebral fracture applications: effects of zirconium dioxide content on the material and setting properties.

The application of bone cements for the treatment of vertebral compression fracture requires radiopaque materials for adequate visualization of the flow under fluoroscopy. Besides high radiopacity, it is desirable for the cement to have relatively low viscosity, high compressive strength and appropriate curing parameters. In this study, the properties of novel two-solution bone cements composed of cross-linked poly (methyl methacrylate) PMMA microspheres or nanospheres added to the linear polymer phase were assessed for formulations with increasing concentrations of zirconium dioxide (ZrO(2)). The addition of a cross-linked phase in the standard two-solution formulation (TSBC) was observed to improve the material properties by increasing setting time and decreasing maximum polymerization temperatures and decreasing the initial viscosity in comparison to the standard cement. The properties of three formulations (TSBC, modified two-solution containing cross-linked PMMA microspheres, and nanospheres) were measured for cements prepared at 0%, 5%, 20%, and 30% ZrO(2) and compared to KyphX. Cements prepared with cross-linked particles exhibited significantly higher compressive strength than the standard-two solution cement and KyphX at increasing radiopacifier concentrations. Furthermore, cement viscosity was increased by the addition of increasing concentrations of ZrO(2) in the modified two-solution cements, whereas the maximum polymerization exotherm and setting time of these materials were decreased. This study indicates that the addition of high concentrations of ZrO(2) significantly affects the properties of two-solution cements acting as a reinforcing phase when cross-linked spheres are added. These materials were observed to be suitable for vertebroplasty applications.

Silybin inhibits interleukin-1β-induced production of pro-inflammatory mediators in canine hepatocyte cultures

Hepatocytes are highly susceptible to cytokine stimulation and are fundamental to liver function. We established primary canine hepatocyte cultures to study effects of anti-inflammatory agents with hepatoprotective properties. Hepatocyte cultures were incubated with control media alone, silybin (SB), or the more bioavailable silybin-phosphatidylcholine complex (SPC), followed by activation with interleukin-1 beta (IL-1β; 10 ng/mL). Inflammatory response was measured by prostaglandin E2 (PGE(2) ), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) production and also nuclear factor-kappa B (NF-κB) translocation. Hepatocyte cultures continued production of the phenotypic marker albumin for more than 7 days in culture. IL-1β exposure increased PGE(2) , IL-8, and MCP-1 production, which was paralleled by NF-κB translocation from the cytoplasm to the nucleus. Pretreatment with SB and SPC significantly inhibited IL-1β-induced production of pro-inflammatory markers and attenuated NF-κB nuclear translocation. We demonstrate for the first time that primary canine hepatocyte cultures can be maintained in culture without phenotypic loss. The observation that hepatocyte cultures respond to pro-inflammatory IL-1β activation indicates hepatocytes as primary cellular targets of extrinsic IL-1β. The ability of SB and SPC to inhibit hepatocyte culture activation by IL-1β reinforces the notion of their hepatoprotective effects. Our primary canine hepatocyte culture model facilitates identification of hepatoprotective agents and their mechanism of action.

An ex vivo exothermal and mechanical evaluation of two-solution bone cements in vertebroplasty.

BACKGROUND CONTEXT Previous ex vivo studies showed that the properties of commercial cements modified for use in vertebroplasty are not optimal and are associated with several drawbacks, including high exothermic reaction, low cement viscosity and consequent extravasation, and unpredictable wait time after cement preparation. Additionally, strength and stiffness restoration are controversial varying with the cement type, volume injected, and technique used. PURPOSE To investigate maximum polymerization temperatures and mechanical performance of novel two-solution bone cement (TSBC) modified by the addition of cross-linked poly(methyl methacrylate) nanospheres (η-TSBC) and microspheres (μ-TSBC) in a cadaver vertebroplasty model in comparison to a commercially available cement (KyphX). To study the viability of application of these novel cement formulations in the treatment of vertebral compression fractures. STUDY DESIGN/SETTING Ex vivo biomechanical and exothermal evaluation of TSBCs using cadaveric vertebral bodies (VBs). METHODS Thirty-one cadaveric vertebrae (age, 74±2 years; T score, -1.5±0.5) were disarticulated. Thirteen vertebrae were assigned into three groups and instrumented with thermocouples positioned midbody along the intersection of the midsagittal and midcoronal axes, as well as along the intersection of the midsagittal axis and posterior VB wall. After equilibration at 37°C, 5 mL of cement was injected and temperatures were recorded for 1 hour. The groups were injected with η-TSBC, μ-TSBC, or KyphX. The remaining 18 vertebrae were biomechanically tested. After randomization into three groups, each specimen was fractured in compression and stabilized with 5 mL of each cement type. Each specimen was then retested in axial compression. RESULTS Temperatures in the central region of the vertebrae were significantly lower (p<.05) when injected with η-TSBC (44°C) in comparison to KyphX (75°C) and μ-TSBC (64°C). A significant difference was not detected between the pre- and postcementing strength (p>.05) of the three groups. There was no significant difference between the average values of stiffness among the cements (p>.05), however there was a significant difference between intact and treated stiffness (p<.05). CONCLUSIONS The TSBC cements decreased the local temperature within the VB while providing similar mechanical strength when compared with vertebrae treated with KyphX.

Micropatterned agarose scaffolds covalently modified with collagen for culture of normal and neoplastic hepatocytes.

Anchorage-dependent cells including hepatocytes, the main functional cellular constituent comprising liver tissue, require a substrate for cell adhesion when cultured outside their native tissue. The challenge with hepatocyte culture is that material substrates and designs supporting hepatocyte attachment, phenotype, and function are not readily available. Our laboratory previously published that type I collagen found in the liver extracellular matrix supports hepatocyte culture. We hypothesized that micropatterned agarose with a coating of collagen covalently bound to the surface would facilitate hepatocyte adhesion and phenotype. To test this hypothesis, primary canine hepatocytes and neoplastic human HepG2 hepatocellular carcinoma cells were cultured on these substrates. Hepatocyte adhesion was dependent on the cell type and also the micropattern design. Viable normal and neoplastic hepatocytes attached to the microchannel troughs rather than on the ridges. In contrast, hepatocyte adhesion on the microcircular patterns was similar to control agarose as cells did not sense differences in surface topology on these substrates. Neoplastic cells exhibited a distinct difference in growth behavior following 7 days in culture on the microchannel patterns, exhibiting aberrant proliferation relative to normal hepatocytes which did not proliferate. Our results suggest that patterned microchannel agarose may be useful to evaluate hepatoprotective and noxious agents.

Hepatoprotective effects of S-adenosylmethionine and silybin on canine hepatocytes in vitro

Inflammation and oxidative stress are associated with liver injury and development of liver disease. The transcription factors nuclear factor-kappa beta (NF-κB) and nuclear factor erythroid 2-related factor 2 (Nrf2) play critical roles in modulating liver injury and damage. Activation of NF-κB induces production of pro-inflammatory molecules including prostaglandin E2 (PGE2 ), interleukin-8 (IL-8) and macrophage chemotactic protein-1 (MCP-1). Nrf2 regulates genes controlling antioxidants. Our laboratory previously showed that hepatocytes, the primary functional cell type comprising liver tissue, respond to the cytokine interleukin-1 beta (IL-1β) by increased production of PGE2 , IL-8 and MCP-1. This increase is associated with nuclear translocation of NF-κB. In this study, we evaluated whether primary canine hepatocytes pre-treated with the combination of S-adenosylmethionine (SAMe; 30 and 2000 ng/ml) and silybin (SB; 298 ng/ml), agents with known anti-inflammatory and antioxidant properties, could attenuate IL-1β-induced inflammation and oxidative stress. The SAMe and SB combination reduced cytokine-induced PGE2 , IL-8 and MCP-1 production while also inhibiting NF-κB nuclear translocation. These changes were accompanied by increased antioxidant enzyme-reduced glutathione (GSH) comparable to control levels. The study shows for the first time that the SAMe and SB combination inhibits both inflammation and oxidative stress through two separate signalling pathways.

PMMA brush-containing two-solution bone cement: preparation, characterization, and influence of composition on cement properties.

Two-solution bone cement consisting of poly (methyl methacrylate) (PMMA) brushes in methyl methacrylate has been developed as an alternative to the traditional two-solution (TSBC) and powder-liquid cements. It was hypothesized that the substitution of brushes, for the entire pre-polymer phase of the cement, would permit a decrease in solution viscosity at higher polymer fractions, and allow for physical entanglements with the cement matrix. Consequently, improved cement exothermal and mechanical properties could be expected with brush addition. PMMA brushes were grafted on the surface of cross-linked PMMA nanospheres following a multi-stage synthetic strategy. Brushes exhibiting optimal molecular weight for preparation of TSBC were used for characterization of cement viscosity, flexural and compressive mechanical properties, exothermal properties and residual monomer content. Interactions between grafts and free polymer formed during free radical polymerization of the cement were evaluated based on molecular weight measurements of the cement matrix and brushes. Brush-containing cements exhibited lower viscosity at significantly higher polymer fractions in comparison to TSBC. Cements with PMMA brushes had significantly lower polymerization temperatures and residual monomer content. Measurements of molecular weight revealed the existence of a dry brush regime when using the brush compositions selected in this study, which led to a reduction in the mechanical properties of some of the compositions tested. The optimal cement viscosity and maintenance of other important cement properties achieved with addition of PMMA brushes is expected to expand the use of the two-solution cements in a range of applications.

Near infrared Raman spectroscopic study of reactive gliosis and the glial scar in injured rat spinal cords

Comparative Raman spectra of ex vivo, saline-perfused, injured and healthy rat spinal cord as well as experiments using enzymatic digestion suggest that proteoglycan over expression may be observable in injured tissue. Comparison with authentic materials in vitro suggest the occurrence of side reactions between products of cord digestion with chondroitinase (cABC) that produce lactones and similar species with distinct Raman features that are often not overlapped with Raman features from other chemical species. Since the glial scar is thought to be a biochemical and physical barrier to nerve regeneration, this observation suggests the possibility of using near infrared Raman spectroscopy to study disease progression and explore potential treatments ex vivo and if potential treatments can be designed, perhaps to monitor potential remedial treatments within the spinal cord in vivo.