Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

University of Akron

The study of single cell mechanics offers a valuable tool for understanding cellular milieus. Specific knowledge of chondrocyte biomechanics could lead to elucidation of disease etiologies and the biomechanical factors most critical to stimulating regenerative processes in articular cartilage. Recent studies in our laboratory have suggested that it may be acceptable to approximate the shape of a single chondrocyte as a disc. This geometry is easily utilized for generating models of unconfined compression. In this study, three continuum mechanics models of increasing complexity were formulated and used to fit unconfined compression creep data. Creep curves were obtained from middle/deep zone chondrocytes (n = 15) and separately fit using the three continuum models. The linear elastic solid model yielded a Young's modulus of 2.55+/-0.85 kPa. The viscoelastic model (adapted from the Kelvin model) generated an instantaneous modulus of 2.47+/-0.85 kPa, a relaxed modulus of 1.48+/-0.35 kPa, and an apparent viscosity of 1.92+/-1.80 kPa-s. Finally, a linear biphasic model produced an aggregate modulus of 2.58+/-0.87 kPa, a permeability of 2.57 x 10(-12)+/-3.09 m(4)/N-s, and a Poisson's ratio of 0.069+/-0.021. The results of this study demonstrate that similar values for the cell modulus can be obtained from three models of increasing complexity. The elastic model provides an easy method for determining the cell modulus, however, the viscoelastic and biphasic models generate additional material properties that are important for characterizing the transient response of compressed chondrocytes.

Unconfined creep compression of chondrocytes.

Although previous studies in the field of tissue engineering have provided important information about articular cartilage, their conclusions are based on population averages and do not account for variations in cell subpopulations. To obtain a precise understanding of chondrocytes, we investigated the effects of cartilage zone and seeding duration on single chondrocyte gene expression to select an optimal zone for tissue engineering (Phase I), followed by an evaluation of growth factor exposure on the zone selected in Phase I (Phase II). In Phase I, superficial and middle/deep bovine articular chondrocytes were seeded in monolayers for 3 or 18 h. In Phase II, middle/deep chondrocytes (selected in Phase I) received 100 ng/ml insulin-like growth factor-I (IGF-I) for 3 h. Real-time reverse transcription/polymerase chain reaction was used to quantify the abundance of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the relative abundances of aggrecan, collagens I and II, cartilage oligomeric matrix protein (COMP), matrix metalloproteinase-1 (MMP-1), and tissue inhibitor of metalloproteinase-1 (TIMP-1). GAPDH varied zonally, but neither time nor IGF-I had an effect on it, suggesting that GAPDH is a suitable housekeeping gene for comparisons within each zone, but not across zones. IGF-I increased the expression of aggrecan and collagen II in middle/deep chondrocytes seeded for 18 h. TIMP-1 expression increased with time in control cells, suggesting that chondrocytes enter a matrix protective state after seeding. IGF-I diminished this effect, suggesting that treatment with IGF-I refocuses chondrocytes on matrix production rather than on protection from metalloproteinases. Concomitant to increasing TIMP-1, MMP-1 was detectable by 18 h in superficial cells, providing further evidence of a trend toward matrix degradation with time. Collagen I was undetected in all cells, and no differences were observed for COMP, confirming that no dedifferentiation or osteoarthritic changes occurred. Taken together, these results establish a unique understanding of individual chondrocyte behavior.

Gene expression of single articular chondrocytes.

Previous work has established that mechanical forces can lead to quantifiable alterations in cell function. However, how forces change gene expression in a single cell and the mechanisms of force transmission to the nucleus are poorly understood. Here we demonstrate that the gene expression of proteins related to the extracellular matrix in single articular chondrocytes is modified by compressive forces in a dosage-dependent manner. Increasing force exposure catabolically shifts single-cell mRNA levels of aggrecan, collagen IIa, and tissue inhibitor of metalloproteinase-1. Cytohistochemistry reveals that the majority of strain experienced by the cell is also experienced by the nucleus, resulting in considerable changes in nuclear volume and structure. Transforming growth factor-beta1 and insulin-like growth factor-I offer mechanoprotection and recovery of gene expression of aggrecan and metalloproteinase-1. These results suggest that forces directly influence gene transcription and may do so by changing chromatin conformation.

Static compression of single chondrocytes catabolically modifies single-cell gene expression.

Stem cell transplantation provides significant promise to regenerative strategies after injury in the central nervous system. Neural stem/progenitor cells (NSPCs) have been studied in terms of their regenerative capacity and their ability to differentiate into neurons when exposed to various soluble factors. In this study, interferon-gamma (IFN-gamma) was compared with brain-derived neurotrophic factor (BDNF) and erythropoietin and was shown to be the best single growth factor for inducing neuronal differentiation from adult rat brain-derived NSPCs. Next, IFN-gamma was surface immobilized to a methacrylamide chitosan (MAC) scaffold that was specifically designed to match the modulus of brain tissue and neuronal differentiation of NSPCs was examined in vitro by immunohistochemistry. Bioactive IFN-gamma was successfully immobilized and quantified by ELISA. Both soluble and immobilized IFN-gamma on MAC surfaces showed dose dependent neuronal differentiation with soluble saturation occurring at 100 ng/mL and the most effective immobilized IFN-gamma dose at 37.5 ng/cm(2), where significantly more neurons resulted compared with controls including soluble IFN-gamma.

Functional immobilization of interferon-gamma induces neuronal differentiation of neural stem cells.

Adult stem cells reside in unique niches that provide vital cues for their survival, self-renewal and differentiation. In order to better understand the contribution of substrate stiffness to neural stem/progenitor cell (NSPC) differentiation and proliferation, a photopolymerizable methacrylamide chitosan (MAC) biomaterial was developed. Photopolymerizable MAC is particularly compelling for the study of the central nervous system stem cell niche because Young's elastic modulus (E(Y)) can be tuned from less than 1 kPa to greater than 30 kPa. Additionally, the numerous free amine functional groups enable inclusion of biochemical signaling molecules that, together with the mechanical environment, influence cell behavior. Herein, NSPCs proliferated on MAC substrates with Young's elastic moduli below 10 kPa and exhibited maximal proliferation on 3.5 kPa surfaces. Neuronal differentiation was favored on the soft est surfaces with E(Y) < 1 kPa as confirmed by both immunohistochemistry and qRT-PCR. Oligodendrocyte differentiation was favored on stiffer scaffolds (> 7 kPa); however, myelin oligodendrocyte glycoprotein (MOG) gene expression suggested that oligodendrocyte maturation and myelination was best on < 1 kPa scaffolds where more mature neurons were present. Astrocyte differentiation was only observed on < 1 and 3.5 kPa surfaces and represented less than 2% of the total cell population. This work demonstrates the importance of substrate stiffness to the proliferation and differentiation of adult NSPCs and highlights the importance of mechanical properties to the success of scaffolds designed to engineer central nervous system tissue.

The effect of substrate stiffness on adult neural stem cell behavior.

The adult central nervous system (CNS) contains adult neural stem/progenitor cells (NSPCs) that possess the ability to differentiate into the primary cell types found in the CNS and to regenerate lost or damaged tissue. The ability to specifically and spatially control differentiation is vital to enable cell-based CNS regenerative strategies. Here we describe the development of a protein-biomaterial system that allows rapid, stable and homogenous linking of a growth factor to a photocrosslinkable material. A bioactive recombinant fusion protein incorporating pro-neural rat interferon-γ (rIFN-γ) and the AviTag for biotinylation was successfully expressed in Escherichia coli and purified. The photocrosslinkable biopolymer, methacrylamide chitosan (MAC), was thiolated, allowing conjugation of maleimide-strepatavidin via Michael-type addition. We demonstrated that biotin-rIFN-γ binds specifically to MAC-streptavidin in stoichiometric yields at 100 and 200 ng/mL in photocrosslinked hydrogels. For cell studies, NSPCs were photo-encapsulated in 100 ng/mL biotin-rIFN-γ immobilized MAC based scaffolds and compared to similar NSPC-seeded scaffolds combining 100 ng/mL soluble biotin-rIFN-γ vs. no growth factor. Cells were cultured for 8 days after which differentiation was assayed using immunohistochemistry for lineage specific markers. Quantification showed that immobilized biotin-rIFN-γ promoted neuronal differentiation (72.8 ± 16.0%) similar to soluble biotin-rIFN-γ (71.8 ± 13.2%). The percentage of nestin-positive (stem/progenitor) cells as well as RIP-positive (oligodendrocyte) cells were significantly higher in scaffolds with soluble vs. immobilized biotin-rIFN-γ suggesting that 3-D immobilization results in a more committed lineage specification.

Differentiation of neural stem cells in three-dimensional growth factor-immobilized chitosan hydrogel scaffolds.

There are currently no acceptable cures for central nervous system injuries, and damage induced large gaps in the peripheral nervous system have been challenging to bridge to restore neural functionality. Innervation by neurons is made possible by the growth cone. This dynamic structure is unique to neurons, and can directly sense physical and chemical activity in its environment, utilizing these cues to propel axons to precisely reach their targets. Guidance can occur through chemoattractive factors such as neurotrophins and netrins, chemorepulsive agents like semaphorins and slits, or contact-mediated molecules such as ephrins and those located in the extracellular matrix. The understanding of biomolecular activity during nervous system development and injury has generated new techniques and tactics for improving and restoring function to the nervous system after injury. This review will focus on the major neuronal guidance molecules and their utility in current tissue engineering and neural regenerative strategies.

Neural regenerative strategies incorporating biomolecular axon guidance signals.

In this study a series of novel, biocompatible hydrogels able to repeatedly takeup and deliver oxygen at beneficial levels have been developed by conjugating various perfluorocarbon (PFC) chains to methacrylamide chitosan via Schiff base nucleophilic substitution, followed by photopolymerization to form hydrogels. The synthesized fluorinated methacrylamide chitosan (MACF) hydrogels were confirmed by high resolution (19)F NMR. Synthesized MACF hydrogels were tested for their ability to takeup and then release oxygen for future use in dermal wound healing. Depending on the PFC substitution type maximum O(2) uptake was observed within 2-6h, followed by complete release to the surrounding environment (5% CO(2)) within 12-120h at oxygen partial pressures of 1-25mm Hg h(-1), providing outstanding system tuning for wound healing and regenerative medicine. MACFs with the most fluorines per substitution showed the greatest uptake and release of oxygen. Interestingly, adding PFC chains with a fluorinated aromatic group considerably enhanced oxygen uptake and extended release compared with a linear PFC chain with the same number of fluorine molecules. MACF hydrogels proved to be readily reloaded with oxygen once release was complete, and regeneration could be performed as long as the hydrogel was intact. Fibroblasts were cultured on MACFs and assays confirmed that materials containing more fluorines per substitution supported the most cells with the greatest metabolic activity. This result was true, even without oxygenation, suggesting PFC-facilitated oxygen diffusion from the culture medium. Finally, MACF gradient hydrogels were created, demonstrating that these materials can control oxygen levels on a spatial scale of millimeters and greatly enhance cellular proliferative and metabolic responses.

Fluorinated methacrylamide chitosan hydrogel systems as adaptable oxygen carriers for wound healing.

Neural stem/progenitor cells (NSPCs) are the stem cell of the adult central nervous system (CNS). These cells are able to differentiate into the major cell types found in the CNS (neurons, oligodendrocytes, astrocytes), thus NSPCs are the mechanism by which the adult CNS could potentially regenerate after injury or disorder. Microenviromental factors are critical for guiding NSPC differentiation and are thus important for neural tissue engineering. In this study, D-mannitol crystals were mixed with photocrosslinkable methacrylamide chitosan (MAC) as a porogen to enhance pore size during hydrogel formation. D-mannitol was admixed to MAC at 5, 10 and 20 wt% D-mannitol per total initial hydrogel weight. D-mannitol crystals were observed to dissolve and leave the scaffold within 1 hr. Quantification of resulting average pore sizes showed that D-mannitol addition resulted in larger average pore size (5 wt%, 4060±160 µm(2), 10 wt%, 6330±1160 µm(2), 20 wt%, 7600±1550 µm(2)) compared with controls (0 wt%, 3150±220 µm(2)). Oxygen diffusion studies demonstrated that larger average pore area resulted in enhanced oxygen diffusion through scaffolds. Finally, the differentiation responses of NSPCs to phenotypic differentiation conditions were studied for neurons, astrocytes and oligodendrocytes in hydrogels of varied porosity over 14 d. Quantification of total cell numbers at day 7 and 14, showed that cell numbers decreased with increased porosity and over the length of the culture. At day 14 immunohistochemistry quantification for primary cell types demonstrated significant differentiation to the desired cells types, and that total percentages of each cell type was greatest when scaffolds were more porous. These results suggest that larger pore sizes in MAC hydrogels effectively promote NSPC 3D differentiation.

3D differentiation of neural stem cells in macroporous photopolymerizable hydrogel scaffolds.

In this study we report the successful synthesis of N-(2-mercaptoethyl)-3-(3-methyl-3H-diazirine-3-yl) propanamide (N-MCEP-diazirine), with sulfhydryl and amine photoreactive ends to allow recombinant protein tethering to chitosan films. This regimen allows mimicry of the physiological endeavor of axon pathfinding in the nervous system where neurons rely on cues for guidance during development and regeneration. Our strategy incorporates strong covalent and noncovalent interactions, utilizing N-MCEP-diazirine, maleimide-streptavidin complex, and two custom biotinylated-fusion proteins, nerve growth factor (bNGF), and semaphorin3A (bSema3A). Synthetic yield of N-MCEP-diazirine was 87.3 ± 1.9%. Characteristic absorbance decrease at 348 nm after N-MCEP-diazirine exposure to UV validated the photochemical properties of the diazirine moiety, and the attachment of cross-linker to chitosan films was verified with Fourier transform infrared spectroscopy (FTIR). Fluorescence techniques showed no significant difference in the detection of immobilized proteins compared to absorbing the proteins to films (p < 0.05); however, in vitro outgrowth of dorsal root ganglia (DRG) was more responsive to immobilized bNGF and bSema3A compared to adsorbed bNGF and bSema3A over a 5 day period. Immobilized bNGF significantly increased DRG length over time (p < 0.0001), but adsorbed bNGF did not increase in axon extension from day 1 to day 5 (p = 0.4476). Immobilized bSema3A showed a significant decrease in neurite length (524.42 ± 57.31 μm) at day 5 compared to adsorbed bSema3A (969.13 ± 57.31 μm). These results demonstrate the superiority of our immobilization approach to protein adsorption because biotinylated-fusion proteins maintain their active confirmation and their tethering can be spatially controlled via a UV activated N-MCEP-diazirine cross-linker.

Specific immobilization of biotinylated fusion proteins NGF and Sema3A utilizing a photo-cross-linkable diazirine compound for controlling neurite extension.

Neural stem/progenitor cells (NSPCs) are able to differentiate into the primary cell types (neurons, oligodendrocytes and astrocytes) of the adult nervous system. This attractive property of NSPCs offers a potential solution for neural regeneration. 3D implantable scaffolds should mimic the microstructure and dynamic properties found in vivo, enabling the natural exchange of oxygen, nutrients, and growth factors for cell survival and differentiation. We have previously reported a new class of materials consisting of perfluorocarbons (PFCs) conjugated to methacrylamide chitosan (MAC), which possess the ability to repeatedly take-up and release oxygen at beneficial levels for favorable cell metabolism and proliferation. In this study, the neuronal differentiation responses of NSPCs to fluorinated methacrylamide chitosan (MACF) hydrogels were studied for 8 days. Two treatments, with oxygen reloading or without oxygen reloading, were performed during culture. Oxygen concentration distributions within cell-seeded MACF hydrogels were found to have higher concentrations of oxygen at the edge of the hydrogels and less severe drops in O2 gradient as compared with MAC hydrogel controls. Total cell number was enhanced in MACF hydrogels as the number of conjugated fluorines via PFC substitution increased. Additionally, all MACF hydrogels supported significantly more cells than MAC controls (p < 0.001). At day 8, MACF hydrogels displayed significantly greater neuronal differentiation than MAC controls (p = 0.001), and among MACF groups methacrylamide chitosan with 15 fluorines per addition (MAC(Ali15)F) demonstrated the best ability to promote NSPC differentiation.

Encapsulated Neural Stem Cell Neuronal Differentiation in Fluorinated Methacrylamide Chitosan Hydrogels.

New developments in growth factor and pharmaceutical control of cells for neural regenerative strategies have built a need for high throughput assays to conveniently screen these treatments in vitro before moving to more complex experiments. Towards this application we have studied an easy and highly reproducible culture regime with a renewable cell source for central nervous system (CNS) strategies. Adult neural stem cells (NSCs) derived from the CNS are attractive for use in screening assay because they are easily expanded and can differentiate into all major CNS cell types. NSCs were cultured on glass alone or methacrylamide chitosan (MAC) hydrogel coated glass substrates, immobilized with extracellular matrix (ECM) proteins collagen and laminin at varying densities. Proteins were also adsorbed on the surfaces as a control. We found that adsorbed protein on hydrogel coated glass resulted in the highest cell densities after 8 d, over twice the density of immobilized groups or adsorbed protein on glass. No significant differences were observed between collagen, laminin, or both proteins together regarding cell differentiation (p > 0.05); however, the morphological spreading and branching of differentiated NSC processes was enhanced on MAC substrates with covalently immobilized laminin protein. The results of this work suggest that a soft MAC hydrogel surface with adsorbed protein would be most desirable for specifying neuronal differentiation of large numbers of stem cells due to the high cellularities they supported.

Nic D. Leipzig

Department of Chemical and Biomolecular Engineering

University of Akron

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Nic D. Leipzig

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Chemical and Biomolecular Engineering

University of Akron

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Department of Chemical and Biomolecular Engineering