Osteoblast response to PLGA tissue engineering scaffolds with PEO modified surface chemistries and demonstration of patterned cell response

Because tissues are characterized by a well-defined three-dimensional arrangement of cells, tissue engineering scaffolds that facilitate the organization and differentiation of new tissue will have improved performance in comparison to scaffolds that only provide surfaces for cell attachment and growth. We hypothesize that instructions for cells can be incorporated into tissue engineering scaffolds by patterning the scaffold's architecture and surface chemistry. Our goals for the presented work were to collect data about cell response to three-dimensional, porous scaffolds with uniformly modified surfaces chemistries, and to demonstrate patterning of cell response by patterning surface chemistry. Our system was osteoblast response to poly(l-lactide-co-glycolide) scaffolds modified with poly(ethylene oxide) (PEO). Scaffolds were fabricated using the Three-Dimensional Printing (3DP) process which has control over scaffolds properties to a resolution of approximately 100 microm in all three dimensions. At higher PEO concentrations, adhesion, growth rates, and migration of rat osteoblasts were reduced; alkaline phosphate activity was increased, and cells were less spread and had microvilli. Patterned regions of low and high cell adhesion were demonstrated on scaffolds fabricated with 1 mm thick stripes of PEO and non-PEO regions.     

Primary cortical glial reaction versus secondary thalamic glial response in the excitotoxically injured young brain: astroglial response and metallothionein expression.

In this study we have evaluated the primary astroglial reactivity to an injection of N-methyl-D-aspartate into the right sensorimotor cortex, as well as the secondary astroglial response in the thalamic ventrobasal complex, caused by the anterograde degeneration of descending corticothalamic fibres and/or target deprivation of the developing thalamic neurons. The astroglial response was evaluated from 4 h to 30 days post-lesion, by the immunocytochemical detection of the cytoskeletal proteins glial fibrillary acidic protein and vimentin, and the antioxidant and metal binding protein metallothionein I-II. In the lesioned cortex, hypertrophied reactive astrocytes showed increased glial fibrillary acidic protein labelling that correlated with a strong expression of vimentin and metallothionein I-II. Maximal astrocytic response was seen at one week post-lesion. The glial scar that formed later on remained positive for all astroglial markers until the last survival time examined. In contrast, in the anterogradely/retrogradely affected thalamus, the induced astroglial secondary response was not as prominent as in the cortex and was characteristically transitory, being undetectable by 14 days post-lesion. Interestingly, thalamic reactive astrocytes showed increased glial fibrillary acidic protein expression but no induction of vimentin and metallothionein I-II. In conclusion, in the young brain, the pattern of astroglial reactivity is not homogeneous and is strongly dependent on the grade of tissue damage: both in response to primary neuronal death and in response to retrograde/anterograde secondary damage, reactive astrocytes show hypertrophy and increased glial fibrillary acidic protein expression. However, astroglial vimentin and metallothionein I-II expression are only observed in areas undergoing massive neuronal death, where glial scar is formed.     

The use of hydrogel microparticles to sequester and concentrate bacterial antigens in a urine test for Lyme disease

Hydrogel biomarker capturing microparticles were evaluated as a biomaterial to amplify the sensitivity of urine testing for infectious disease proteins. Lyme disease is a bacterial infection transmitted by ticks. Early diagnosis and prompt treatment of Lyme disease reduces complications including arthritis and cardiac involvement. While a urine test is highly desirable for Lyme disease screening, this has been difficult to accomplish because the antigen is present at extremely low concentrations, below the detection limit of clinical immunoassays. N-isopropylacrylamide (NIPAm)-acrylic acid (AAc) microparticles were covalently functionalized with amine containing dyes via amidation of carboxylic groups present in the microparticles. The dyes act as affinity baits towards protein analytes in solution. NIPAm/AAc microparticles functionalized with acid black 48 (AB48) mixed with human urine, achieved close to one hundred percent capture and 100 percent extraction yield of the target antigen. In urine, microparticles sequestered and concentrated Lyme disease antigens 100 fold, compared to the absence of microparticles, achieving an immunoassay detection sensitivity of 700 pg/mL in 10 mL urine. Antigen present in a single infected tick could be readily detected following microparticle sequestration. Hydrogel microparticles functionalized with high affinity baits can dramatically increase the sensitivity of urinary antigen tests for infectious diseases such as Lyme disease. These findings justify controlled clinical studies evaluating the sensitivity and precision of Lyme antigen testing in urine.     

Role of Neurotrophic Factors BDNF and GDNF in Nervous System Adaptation to the Influence of Ischemic Factors

Bulletin of Experimental Biology and Medicine - C57Bl/6J mice were exposed to γ-radiation in a dose of 7.5 Gy. A week later, the experimental group received intravenous injection of...     

NGF receptor immunoreactivity in rat brain: topographic distribution and response to entorhinal ablation

Nerve growth factor receptor (NGFR) immunoreactivity was distributed in neuronal cell bodies, axons and/or dendrites of cholinergic and non-cholinergic structures of the rat brain. The dentate gyrus of normal animals showed immunostained fibers mainly in the supragranular band, whereas following entorhinal ablation a greater density of immunostained fibers appeared in the outer molecular layer and a wider clear zone was apparent in the inner molecular layer. The NGFR-positive fibers followed the pattern of reorganization of septal fibers visualized with acetylcholinesterase staining. The results suggest that the NGF/NGFR may have a role in reorganization of septal fibers following entohinal lesion.     

Time course study of the antigen-specific immune response to a PLGA microparticle vaccine formulation

Microparticle-based vaccine delivery systems are known to promote enhanced immune responses to protein antigens and can elicit T_H1-biased responses when used in combination with Toll-like receptor (TLR) agonists. It is important to understand the kinetics of the immune responses to microparticle-based protein vaccines in order to predict the duration of protective immunity and to optimize prime-boost vaccination regimens. We carried out a 10-week time course study to investigate the magnitude and kinetics of the antibody and cellular immune responses to poly(lactic-co-glycolic acid) (PLGA) microparticles containing 40 μg ovalbumin (OVA) protein and 16 μg CpG-ODN adjuvant (MP/OVA/CpG) in comparison to OVA-containing microparticles, soluble OVA plus CpG, or OVA formulated with Alhydrogel^® aluminum adjuvant. Mice vaccinated with MP/OVA/CpG developed the highest T_H1-associated IgG2b and IgG2c antibody titers, while also eliciting T_H2-associated IgG1 antibody titers on par with Alhydrogel^®-formulated OVA, with all IgG subtype titers peaking at day 56. The MP/OVA/CpG vaccine also induced the highest antigen-specific splenocyte IFN-γ responses, with high levels of IFN-γ responses persisting until day 42. Thus the MP/OVA/CpG formulation produced a sustained and heightened humoral and cellular immune response, with an overall T_H1 bias, while maintaining high levels of IgG1 antibody equivalent to that seen with Alhydrogel^® adjuvant. The time course kinetics study provides a useful baseline for designing vaccination regimens for microparticle-based protein vaccines.     

Primary cortical glial reaction versus secondary thalamic glial response in the excitotoxically injured young brain: microglial/macrophage response and major histocompatibility complex class I and II expression

The excitatory amino acid analog, N-methyl-D-aspartate, was injected intracortically into nine-day-old rats. Resulting axon-sparing lesions in the developing sensorimotor cortex, which secondarily affect thalamic neurons that become deprived of cortical targets, provide an experimental model for the study of the glial response in distantly affected areas. The microglial/macrophage response was studied using tomato lectin histochemistry and major histocompatibility complex I and II immunocytochemistry. Blood-brain barrier integrity was evaluated. In the cortical lesion site, where blood-brain barrier breakdown occurs, the rapid microglial response was restricted to the degenerating area. Microglial changes were first seen at 4 h post-injection, peaking at days 3-5. Reactive microglia changed morphology, increased tomato lectin binding and expressed major histocompatibility complex I. Additionally, some cells expressed major histocompatibility complex II. In the secondarily affected thalamus, the microglial response was not as pronounced as in the cortex, was first seen at 10 h post-injection and peaked at days 3-5. Reactive microglia showed a bushy morphology, were intensely lectin positive and expressed major histocompatibility complex I. The exceptional response of the nine-day-old brain to cortical lesions makes this model an interesting tool for studying the implications of microglial major histocompatibility factor expression in still enigmatic processes such as wound healing and plasticity.     

Protein deimination in the rat brain after kainate administration: citrulline-containing proteins as a novel marker of neurodegeneration

Peptidylarginine deiminases (PADs) are a group of enzymes that convert protein arginine residues to citrulline residues in a Ca2+-dependent manner. In the central nervous system, PAD type II localizes in glial cells, but its biological role is little understood. We examined the timing and region dependence of protein deimination in the rat cerebrum after a systemic injection of kainic acid (KA). Citrulline-containing proteins were consistently found in neurodegenerating regions. Western blot analyses showed deimination of numerous proteins in a broad-molecular-weight range. By immunocytochemical scrutiny, deiminated protein-positive astrocytes were found at 2 h after KA administration, and they increased in number until the 6 h. Furthermore, shrunken neurons became deiminated protein-positive at 12-24 h. These data suggest that PAD type II becomes activated in regions undergoing neurodegeneration and functions to deiminate various proteins. Therefore, citrulline-containing proteins seem to be a useful marker of acute neurodegeneration.     

The distribution of GABA and glycine response in the mouse brain using Xenopus oocytes

Injection of mRNA from different regions of the central nervous system (CNS) of the mouse into the Xenopus oocyte caused a difference in the relative glycine to gamma-aminobutyric acid (GABA) response of the oocyte. Glycine caused the response in oocytes injected with brainstem mRNA but hardly in oocytes injected with cerebrum mRNA and in oocytes injected with cerebellum mRNA. In contrast, GABA caused the response in oocytes injected with mRNA from each of the 3 parts of the CNS. These obvious regional differences may reflect the distribution of the receptors to each plausible neurotransmitter in the CNS.     

Genetic variants in the BDNF gene and therapeutic response to risperidone in schizophrenia patients: a pharmacogenetic study

Risperidone is a widely used atypical antipsychotic agent that produces considerable interindividual differences in patient response. We investigated the pharmacogenetic relationship between the brain-derived neurotrophic factor (BDNF) gene and response to risperidone in 127 Han Chinese schizophrenic patients. Three functional polymorphisms, (GT)_n dinucleotide repeat polymorphism, C-270T, and the rs6265G/A single-nucleotide polymorphism (SNP), were genotyped and analyzed for association, with reduction of Brief Psychiatric Rating Scale (BPRS) scores following an 8-week period of risperidone monotherapy. For individual polymorphic analysis, we found that the frequency of the 230-bp allele of the (GT)_n polymorphism was much higher in responders (47.95%) than in nonresponders (32.41%) and the difference was statistically significant even after Bonferroni''s adjustment (for the 230-bp allele: adjusted P=0.039). For haplotype-based analyses of the three polymorphisms, no positive finding was observed in the global test, but in specific haplotype tests, two haplotypes were also significantly related to response to risperidone (for haplotype 230-bp/C-270/rs6265G: P=0.0009; for haplotype 234-bp/C-270/rs6265A: P=0.043), indicating that patients with the 230-bp allele of the (GT)_n polymorphism or the 230-bp/C-270/rs6265G haplotype responded better to risperidone than those with other alleles or haplotypes, and that the positive effect of the individual haplotype 230-bp/C-270/rs6265G was mainly driven by the 230-bp allele. These findings demonstrate that the individual and combinatorial genetic variants in the BDNF gene might have a role in the therapeutic response to risperidone in the Han Chinese population.     

The physical properties and response of osteoblasts to solution cast films of PLGA doped polycaprolactone

Polycaprolactone films doped with poly(lactide-co-glycolide) (65:35) in 0, 10, 20, and 30 (wt%) were prepared and evaluated in terms of morphology, dynamic contact angle analysis, and thermal properties. The unique surface morphology of the doped PCL film resulted, without introducing significant microstructure disruption of PCL aggregation. The doped PCL film registered a lower contact angle and increased hydrophilicity. Osteoblast cells attached to all doped materials, the 10% and 20% doped materials demonstrating the greatest cell growth.     

HMGB1 and microparticles as mediators of the immune response to cell death

In a wide variety of diseases, cell death represents both an outcome and an important step in pathogenesis. This duality occurs because cell death leads to the extracellular release of molecules and structures that can potently induce the innate immune system. These mediators include the alarmins which are endogenous cellular constituents that exit activated or dying cells to stimulate toll-like receptors (TLRs) as well as non-TLR receptors. Of alarmins, the nonhistone protein HMGB1 is the prototype. Like DNA and RNA, HMGB1 can translocate from cells as they die. The activity of HMGB1 may reflect its interaction with other molecules such as LPS, DNA, and cytokines. In addition to alarmins, dead and dying cells can release subcellular organelles called microparticles that contain cytoplasmic and nuclear constituents, including DNA and RNA. These particles can impact on many cell types to induce inflammation. The release of HMGB1 and microparticles shows important similarities, occurring with cell death as well as stimulation of certain but not all TLRs. Furthermore, nitric oxide can induce the release of both. These observations suggest that the products of dead cells can serve as important mediators to drive immune responses and promote inflammation and autoreactivity.     

Vaccination against nicotine during continued nicotine administration in rats: immunogenicity of the vaccine and effects on nicotine distribution to brain

Vaccination against nicotine has been proposed as a potential treatment for nicotine dependence. Because vaccination may take months to elicit satisfactory antibody levels, the clinical usefulness of this approach will be enhanced if vaccination can be accomplished during continued nicotine intake (e.g., before a smoker quits). The current study examined the immunogenicity of a nicotine conjugate vaccine during continued nicotine dosing in rats, and its effects on nicotine distribution to brain. In the first experiment, nicotine was administered over 11 weeks as 20 intra venous (i.v.) bolus injections per day during the rat's active cycle to simulate the usual pattern of nicotine intake from cigarette smoking. In the second experiment, rats received a continuous s.c. infusion of nicotine by osmotic pump for 11 weeks to provide serum nicotine concentrations equivalent to those of a heavy smoker and 24 h/day nicotine exposure. Nicotine-specific antibody titers after the third booster dose were not compromised by either regimen of concurrent nicotine administration compared to those of rats receiving saline. A single additional i.v. nicotine dose was administered at the end of each experiment. The distribution of this single nicotine dose to brain was reduced by 40-60% in vaccinated rats compared to controls. Vaccine efficacy in reducing nicotine distribution to brain was not compromised by concurrent nicotine administration. These data suggest that vaccination during concurrent nicotine administration is feasible, and that the ability of vaccination to reduce nicotine distribution to brain is preserved even after months of nicotine dosing at rates approximating cigarette smoking.     

The cardiovascular response of normal humans to the administration of endotoxin

Marked abnormalities in cardiovascular function accompany septic shock, and bacterial endotoxin is believed to be one of the principal mediators of these abnormalities. To evaluate the cardiovascular effects of endotoxemia in humans, we measured hemodynamic variables in nine normal subjects given an intravenous bolus dose of endotoxin (Escherichia coli, 4 ng per kilogram of body weight) and in six normal subjects given a bolus dose of saline, before and three hours after administration. All the subjects then underwent volume loading with normal saline (mean, 2217 ml) during the fourth and fifth hours after administration of the bolus, and the measurements were repeated. Three hours after the administration of endotoxin and before volume loading, the cardiac index had increased by 53 percent and the heart rate by 36 percent (both changes were significant; P less than or equal to 0.008), and the systemic vascular-resistance index had decreased by 46 percent (P = 0.004). After volume loading (five hours after the administration of endotoxin), the left ventricular ejection fraction decreased by 1 percent of the base-line value in the subjects given endotoxin, but increased by 14 percent in the controls (P = 0.008). The left ventricular end-diastolic and end-systolic volume indexes increased by 18 percent (P = 0.07) and 24 percent (P = 0.042), respectively. Left ventricular performance, as measured by the ratio of the peak systolic pressure to the end-systolic volume index, was depressed (a decrease of 0.90 in the subjects given endotoxin vs. an increase of 0.76 in the controls; P = 0.024). We conclude that the administration of endotoxin to normal subjects causes a depression of left ventricular function that is independent of changes in left ventricular volume or vascular resistance. The changes in function are similar to those observed in septic shock and suggest that endotoxin is a major mediator of the cardiovascular dysfunction in this condition.     

The glia doctrine: Addressing the role of glial cells in healthy brain ageing

Glial cells in their plurality pervade the human brain and impact on brain structure and function. A principal component of the emerging glial doctrine is the hypothesis that astrocytes, the most abundant type of glial cells, trigger major molecular processes leading to brain ageing. Astrocyte biology has been examined using molecular, biochemical and structural methods, as well as 3D brain imaging in live animals and humans. Exosomes are extracelluar membrane vesicles that facilitate communication between glia, and have significant potential for biomarker discovery and drug delivery. Polymorphisms in DNA repair genes may indirectly influence the structure and function of membrane proteins expressed in glial cells and predispose specific cell subgroups to degeneration. Physical exercise may reduce or retard age-related brain deterioration by a mechanism involving neuro-glial processes. It is most likely that additional information about the distribution, structure and function of glial cells will yield novel insight into human brain ageing. Systematic studies of glia and their functions are expected to eventually lead to earlier detection of ageing-related brain dysfunction and to interventions that could delay, reduce or prevent brain dysfunction.     

Chronic cocaine administration to rats alters the distribution of cell types recorded in vitro within the dorsolateral septal nucleus

 We investigated persistent changes in single-neuron activity in the dorsolateral septal nucleus (DLSN) induced by chronic administration of cocaine. Intracellular recording techniques were utilized with an in vitro brain slice preparation to examine the effects of in vivo chronic cocaine administration, for 7 days or 14 days, on the distribution of electrophysiologically characterized DLSN cells. We have previously distinguished DLSN neurons into three major types (I, II, and III), based upon their action potential configuration and firing pattern. This study demonstrated that type III neurons were over-represented in brain slices obtained from rats treated chronically with cocaine in vivo for 14 days when compared with brain slices obtained from rats treated either with cocaine for only 7 days or with saline and never exposed to cocaine. These data provide further evidence that neurons undergo plastic changes following chronic cocaine administration. Received: 22 August 1996 / Accepted: 21 April 1997     

The regional distribution of d-amphetamine and local glucose utilization in rat brain during continuous amphetamine administration

Summary The distribution of radioactivity following administration of either [³H]d-amphetamine or [³H]2-deoxy-d-glucose was examined by scintillation counting of 22 microdissected brain regions from rats pretreated with either acute or continuous amphetamine, or continuously administered labeled d-amphetamine. Animals continuously administered drug were sacrificed in behaviorally distinct stages of the continuous amphetamine syndrome, a potential animal model of amphetamine psychosis.Both isotopes were heterogeneously distributed within brain, and their distributions were differentially affected by acute or continuous amphetamine regimens. While the distribution of either isotope in naive rats was characterized by greatest concentrations of counts in rostral rather than caudal regions, and grey-matter rather than white-matter structures, continuous amphetamine administration resulted in progressively increased retention of amphetamine by mesolimbic but not nigrostriatal brain regions; this was accompanied by locally enhanced levels of glucose utilization. This effect was predominantly localized in the nucleus accumbens, which exhibited the greatest retention of amphetamine and greatest relative increase in glucose utilization of any region studied during that stage of the continuous amphetamine syndrome thought to best model amphetamine psychosis. Alterations in amphetamine distribution and local levels of neural activity may reflect a change in the principal locus of control of amphetamine effects within brain as animals progress through the stages of the continuous amphetamine syndrome.Supported by U.S.P.H.S. grant DA 02312     

The use of covalently immobilized stem cell factor to selectively affect hematopoietic stem cell activity within a gelatin hydrogel

Hematopoietic stem cells (HSCs) are a rare stem cell population found primarily in the bone marrow and responsible for the production of the body's full complement of blood and immune cells. Used clinically to treat a range of hematopoietic disorders, there is a significant need to identify approaches to selectively expand their numbers ex vivo. Here we describe a methacrylamide-functionalized gelatin (GelMA) hydrogel for in vitro culture of primary murine HSCs. Stem cell factor (SCF) is a critical biomolecular component of native HSC niches in vivo and is used in large dosages in cell culture media for HSC expansion in vitro. We report a photochemistry based approach to covalently immobilize SCF within GelMA hydrogels via acrylate-functionalized polyethylene glycol (PEG) tethers. PEG-functionalized SCF retains the native bioactivity of SCF but can be stably incorporated and retained within the GelMA hydrogel over 7 days. Freshly-isolated murine HSCs cultured in GelMA hydrogels containing covalently-immobilized SCF showed reduced proliferation and improved selectivity for maintaining primitive HSCs. Comparatively, soluble SCF within the GelMA hydrogel network induced increased proliferation of differentiating hematopoietic cells. We used a microfluidic templating approach to create GelMA hydrogels containing gradients of immobilized SCF that locally direct HSC response. Together, we report a biomaterial platform to examine the effect of the local presentation of soluble vs. matrix-immobilized biomolecular signals on HSC expansion and lineage specification. This approach may be a critical component of a biomaterial-based artificial bone marrow to provide the correct sequence of niche signals to grow HSCs in the laboratory.     

The binary complex of poly(PEGMA-co-MAA) hydrogel and PLGA nanoparticles as a novel oral drug delivery system for ibuprofen delivery

To improve the bioavailability of ibuprofen (IBU), we developed a novel binary complex of poly(PEGMA-co-MAA) hydrogel and IBU-loaded PLGA nanoparticles (IBU-PLGA NPs@hydrogels) as an oral intestinal targeting drug delivery system (OIDDS). The IBU-loaded PLGA NPs and pH-sensitive hydrogels were obtained via the solvent evaporation method and radical polymerization, respectively. The final OIDDS was obtained by immersing the hydrogel chips in the IBU-loaded PLGA NPs solutions (pH 7.4) for 3 d. The size distribution and morphology of cargo-free NPs were studied by laser granularity analyzer and transmission electron microscope (TEM). The inner structures of the pH-sensitive hydrogel chips were observed with an S-4800 scanning electron microscope (SEM). The distribution states of IBU in the OIDDS were also studied with X-ray diffraction (XRD) and differential scanning calorimetry (DSC). TEM photographs illustrated that the PLGA NPs had a round shape with an average diameter about 100 nm. Fourier transform infrared spectrum (FTIR) confirmed the synthesis of poly(PEGMA-co-MAA) hydrogel. The SEM picture showed that the final hydrogel had 3D net-work structures. Moreover, the poly(PEGMA-co-MAA) hydrogel showed an excellent pH-sensitivity. The XRD and DSC curves suggested that IBU distributed in the OIDDS with an amorphous state. The cumulated release profiles indicated that the final OIDDS could release IBU in alkaline environment (e.g. intestinal tract) at a sustained manner. Therefore, the novel OIDDS could improve the oral bioavailability of IBU, and had a potential application in drug delivery.