Name: fabrication and characterization of griffithsin-modified fiber scaffolds for prevention of sexually transmitted infections
Uploaded: 2017-10-31T14:00:00
Description: Watch this Scientific Journal Video about Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections at JoVE.com

We are grateful to the Jewish Heritage Fund for Excellence for funding this research. We thank Dr. Stuart Williams II&#160;for generously providing usage of the electrospinning system. We also thank Dr. Kenneth Palmer for providing us with Griffithsin. We additionally thank Dr. Nobuyuki Matoba and his lab for training us in the GRFT ELISA work.

1. Preparation and Fabrication of the Electrospun Fiber Scaffold
CAUTION: All work with solvents or polymer solutions should be performed in a chemical fume hood. Refer to material safety datasheet of each reagent before starting the protocol.
<ol>
	<li>To electrospin a 3 mL 15% w/w PLGA polymer solution, weigh 720 mg of 50:50 poly(lactic-co-glycolic acid) (PLGA; 0.55 to 0.75 dL/g, 31-57 kDa) into a 10 mL scintillation vial. The volume of the solution is based on the typical batch s...

Due to their porous structures and large surface areas, EFs have found a variety of applications in healthcare, one of which includes serving as therapeutic delivery vehicles. Drugs and other active agents can be incorporated within EFs for tunable delivery, while biologics and chemical ligands can be conjugated to the fiber surface for cell-specific targeting52 or biosensing53. Here the fabrication of GRFT surface-modified PLGA EFs, as a delivery scaffold to prevent HIV in...

The use of EFs as a topical delivery platform has the potential to significantly reduce STIs. Currently, there are over 36 million people living with HIV, with over two million new cases of reported in 2015 alone1,2. Additionally, herpes simplex virus type 2 (HSV-2) infection affects hundreds of millions of people worldwide and has been shown to enhance the acquisition of HIV by 2 - 5 fold3. Due to this relationship between HSV-2 infection and HIV acquisition, there is significant interest in developing new active agents that provide simultaneous protection against multiple STIs. Moreover, the development of new vehicles to improve the delivery of these antiviral agents offers the potential to further enhance protective and therapeutic potency. Toward this goal, EFs have been investigated as a new delivery platform to reduce the prevalence of HIV-1 and HSV-2 infections.
During the past two decades, EFs have been extensively used in the fields of drug delivery and tissue engineering4. Often, biocompatible polymers are selected to easily translate to therapeutic applications. To fabricate polymeric EFs, the selected polymer is dissolved in an organic solvent or aqueous solution, depending on the degree of polymer hydrophobicity5. Active agents of interest are then added to the solvent or aqueous solution prior to the electrospinning process. The polymer solution is then aspirated into a syringe and slowly ejected while subject to an electrical current. This process typically results in polymer fibers with sheet or cylindrical macrostructures (Figure 1), and fiber diameters ranging from the micro- to nano-scale6. For most therapeutic applications, active agents are incorporated within the fibers during the electrospinning process and are released from the fiber via diffusion and subsequent fiber degradation. The rate of degradation or release may be altered by using different types of polymers or polymer blends to establish a desired release profile, imparting unique chemical and physical properties7, and promoting the encapsulation of virtually any compound. As such, EFs have proven beneficial to the delivery of small molecule drugs and biological agents including proteins, peptides, oligonucleotides, and growth factors6,8,9.
In the field of STI prevention, EFs have been recently used to incorporate and provide sustained- or inducible-release of antiviral agents10,11,12,13,14,15,16,17,18,19. In one of the earliest studies, pH-responsive fibers were developed to release active agents in response to environmental changes within the female reproductive tract (FRT), as an on-demand method of protection against HIV-111. Since, other studies have investigated polymer blends comprised of polyethylene oxide (PEO) and poly-L-lactic acid (PLLA), to evaluate the tunable release of antiviral and contraceptive agents for HIV-1 prevention and contraception in vitro12. Additional studies have demonstrated the feasibility of EFs to provide the following: prolonged release of small molecule antivirals14, strong and flexible mechanical properties20, 3-D delivery architectures21, inhibition of sperm penetration12, and the ability to merge with other delivery technologies13. Finally, previous work has evaluated polymeric fibers for the sustained-delivery of antiviral agents against common co-infective viruses, HSV-2 and HIV-114. In this study, polymer fibers provided complementary activity to antiviral delivery by retaining their structure for up to 1 month and providing a physical barrier to viral entry. From these results, it was observed that EFs may be used to both physically and chemically hinder virus infection.
While tunable release properties make polymeric EFs an attractive delivery platform for microbicide delivery, EFs have been developed in other applications to serve as surface-modified scaffolds7. EFs have been used to mimic the morphology of the extracellular matrix (ECM), often acting as scaffolds to improve cellular regeneration22, and enhance their utility in tissue engineering23,24. Fibers comprised of polymers such as poly-&#949;-caprolactone (PCL) and PLLA have been surface-modified with growth factors and proteins after electrospinning to impart ECM-like properties including increased cellular adhesion and proliferation25,26. Additionally, antimicrobial surface-modified EFs have been evaluated to prevent the growth of specific pathogenic bacteria27,28. Due to this versatility and the ability to induce biological effects, EF technology continues to expand across a variety of fields to provide multi-mechanistic functionality. Yet, despite their utility in a diversity of applications, surface-modified fibers have only recently been explored in the microbicide field29.
In parallel with the development of new delivery technologies to prevent and treat STIs, novel biological therapeutics have been developed. One of the most promising microbicide candidates is the adhesive antiviral lectin, GRFT30. Originally derived from a species of red algae, GRFT has demonstrated activity as a potent inhibitor of HIV, HSV-2, SARS, as well as Hepatitis C virus31,32,33,34,35,36. In fact, among biologically-based inhibitors, GRFT has the most potent anti-HIV activity, inactivating HIV-1 almost immediately upon contact30, while maintaining stability and activity in the presence of culture media from vaginal microbes for up to 10 days37. More recently, a 0.1% GRFT gel was shown to protect mice against intravaginal HSV-2 challenge, making it a promising candidate for the first line of protection against both HSV-2 and HIV-132,38. For HIV specifically, GRFT inhibits infection by physically binding gp120 or terminal mannose N-linked glycan residues on viral envelope surfaces to prevent entry38,39,40,41,42. This inhibition is highly potent, with IC50s approaching 3 ng/mL43. In addition to inhibiting HIV infection, studies have also shown that GRFT protects against HSV-2 infection by inhibiting the cell-to-cell spread of the virus32. In all cases, GRFT has been shown to be adhesive to viral particles, while demonstrating high resistance to denaturation. Last, GRFT has demonstrated synergistic activity with combinations of Tenofovir (TFV) and other antivirals44, making it feasible and likely beneficial to co-administer with EFs. The potent properties of GRFT make it an excellent biologically-based antiviral candidate, in which delivery may be enhanced with EF technology.
Utilizing this knowledge of the adhesive and innate antiviral properties of GRFT, a polymeric fiber scaffold was designed, that integrates these properties to provide the first layer of virus entry inhibition29. Finding inspiration in the way that cervicovaginal mucus hinders virus transport primarily through mucoadhesive mucin interactions, we hypothesized that by using EFs as a scaffold and covalently modifying the surface with GRFT, a high density of surface-conjugated GRFT would debilitate and inactivate virus at its entrypoint45,46,47. Here EFs were developed as a stationary scaffold to provide a protein-based, viral adhesive-inactivating barrier platform. We sought to combine the potent antiviral properties of GRFT with a biocompatible, modifiable, and durable polymer platform, to create a novel virus &#34;trap.&#34;
To achieve these goals, fibers comprised of PLGA were electrospun, and EDC-NHS chemistry was used to subsequently modify the EF surface with GRFT. PLGA served as a model polymer due to its extensive use in electrospinning48, combined with its biocompatibility and cost-effectiveness. Additionally, surface modification exploits the large surface area of EFs, and provides a useful alternative that can be combined with encapsulation to maximize fiber utility49. Unlike traditional encapsulation methods where only a portion of GRFT is available (and only transiently present in the FRT), surface modification may enable GRFT to maintain maximum bioactivity during the entire duration of treatment. Furthermore, the incorporation of hydrophilic compounds such as proteins, by traditional electrospinning methods, may result in lower encapsulation efficiencies and loss of protein activity50. Therefore, GRFT surface-modified fibers may offer a promising alternative delivery method that can be used alone or in combination with electrospinning to enhance protection against STI infection.

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			<td class="xl21" height="21" style="height:15.75pt">1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP)</td>
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fabrication and characterization of griffithsin-modified fiber scaffolds for prevention of sexually transmitted infections

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to prevent and treat sexually transmitted infections (STIs). Moreover, many EF technologies focus on encapsulating the active agent, relative to utilizing the surface to impart biofunctionality. Here we describe a method to fabricate and surface-modify poly(lactic-co-glycolic) acid (PLGA) electrospun fibers, with the potent antiviral lectin Griffithsin (GRFT). PLGA is an FDA-approved polymer that has been widely used in drug delivery due to its outstanding chemical and biocompatible properties. GRFT is a natural, potent, and safe lectin that possesses broad activity against numerous viruses including human immunodeficiency virus type 1 (HIV-1). When combined, GRFT-modified fibers have demonstrated potent inactivation of HIV-1 in vitro. This manuscript describes the methods to fabricate and characterize GRFT-modified EFs. First, PLGA is electrospun to create a fiber scaffold. Fibers are subsequently surface-modified with GRFT using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS)chemistry. Scanning electron microscopy (SEM) was used to assess the size and morphology of surface-modified formulations. Additionally, a gp120 or hemagglutinin (HA)-based ELISA may be used to quantify the amount of GRFT conjugated to, as well as GRFT desorption from the fiber surface. This protocol can be more widely applied to fabricate fibers that are surface-modified with a variety of different proteins.

Fiber morphology has a significant effect on the ability of surface-modified EFs to provide protection against viruses. Although electrospinning is a convenient and straightforward procedure, non-optimized polymer formulations may result in irregular fiber morphology (Figure 5B-C). Alterations in electrospinning conditions that result in the formation of beaded or amorphous mat-like morphologies, are often caused by solvent-polymer incompatib...

Watch this Scientific Journal Video about Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections at JoVE.com

Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections

This manuscript describes the procedure to fabricate and characterize Griffithsin-modified poly(lactic-co-glycolic acid) electrospun fibers that demonstrate potent adhesive and antiviral activity against human immunodeficiency virus type 1 infection in vitro. Methods used to synthesize, surface-modify, and characterize the resulting morphology, conjugation, and desorption of Griffithsin from surface-modified fibers are described.

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to ...

The overall goal of this procedure is to fabricate and surface-modify polylactic acid, co-glycolic acid, electrospun fibers, with varying amounts of the antiviral protein Griffithsin. This method can help answer key questions in the drug delivery field, such as how to formulate and characterize new delivery vehicles that can prevent sexually transmitted viral infections, such as HIV-1. The main advantage of the technique is that the antiviral activity of Griffithsin is maximized by using polymeric-electrospun fibers as a stationary scaffold to highly localized Griffithsin on the fiber surface.The implications of this technique extend toward therapy of other viral and bacteria infections, because of Griffithsin's potent prophylactic and therapeutic potential against a variety of infection types. Though this method can provide insight into preventing HIV-1 infection, it can also be applied to other systems, such as other viral or bacterial infections in which Griffithsin has demonstrated potency. To begin this procedure, weigh 720 milligrams of 50/50 PLGA into a 10 milliliter scintillation vile.Using a serological glass pipette, add 3.0 milliliters of HFIP. Cover the vile with plastic film. Then, measure and record the vile mass.Incubate the polymer suspension at 37 degrees Celsius overnight to ensure complete dissolution of the polymer. After this, prepare the electrospinning apparatus as outlined in the text protocol. Use a three milliliter syringe to aspirate the polymer solution.Next, connect a blunt 18 gauge 0.5 inch needle tip to the syringe. Dispense the the excess solution to remove empty head space in the needle. Place the syringe on a syringe pump, and set the instrument flow rate to 2.0 milliliters per hour.Connect the power source to the syringe needle. Electrospin the polymer solution using a voltage of positive 27 kilovolts. After the entire solution is electrospun, turn off the power source.Allow the mandril to spin for an additional 30 minutes, to fully evaporate the solvent. Then, turn off the rotating mandril collector. Using a razor blade, gently peel the fiber from the mandril.Collect the electrospun PLGA fiber into a labeled petri dish. Place the dish in a desiccator overnight to remove any residual solvent. To begin, prepare the PBS and MES solutions as outlined in the text protocol.Next, remove the EDC and NHS from the freezer. Let them rest to equilibrate to room temperature. After this, weigh four milligrams of EDC into a 1.5 milliliter micro-centrifuge tube.Weigh six milligrams of NHS into a different micro-centrifuge tube. Add one milliliter of MES buffer to each tube. Vortex both tubes vigorously to ensure the reagents are fully dissolved.Then, weigh 70 milligrams of hydroxylamine into a 50 milliliter conical centrifuge tube. Add 20 milliliters of PBS, and vortex. Mass out an appropriate amount of PLGA fiber into a 15 milliliter conical centrifuge tube.Add eight milliliters of MES buffer to the fiber. Next, add one milliliter of both the EDC and NHS solutions. Use plastic film to seal the 15 milliliter conical centrifuge tube.Place the tube on a rotator to gently invert the solution for 15 minutes at room temperature. After this, add 14 microliters of beta-mercaptoethanol to quench the reaction. Invert the tube several times to ensure complete mixing.Discard the supernatant, and rinse the PLGA fiber twice using 10 milliliters of PBS for each wash. Add an appropriate amount of GRFT stock solution to the tube, as outlined in the text protocol. Then, add enough PBS to bring the final volume to eight milliliters.Close and invert the tube to ensure thorough mixing. Seal the tube with plastic film. Place the tube on the rotator to gently invert the solution for two hours.After this, quench the reaction by adding two milliliters of the prepared hydroxylamine solution, and vortexing for 30 seconds. Discard the supernatant. Rinse the surface modified PLGA fiber twice, using 10 milliliters ultra pure water, and vortexing for each wash.Next, transfer the washed fiber to a petri dish. Place the petri dish inside of a desiccator, until the fiber is completely dry. Then store the petri dish at four degrees Celsius.To begin, set out three 1.5 milliliter micro-centrifuge tubes. Mass out two milligrams of fiber into each. Then, add one milliliter of DMSO to each tube.Vortex for one minute at room temperature to completely dissolve the fiber. After this, dilute a 10 microliter aliquot at least 100 fold, in TE buffer. Store at minus 20 degrees Celsius, until loading characterization with Eliza.Weigh out between 5 and 10 milligrams of surface modified fiber, and transfer it to a micro-centrifuge tube. Record the fiber mass placed in each tube. Next, add one milliliter of a solution that mimics the physiological environment of each sample.Incubate the samples on a rotating shaker at 200 RPM and 37 degrees Celsius for one hour. After this, remove approximately one milliliter of the solution containing the desorbed GRFT and aliquot it into cluster tubes. Store at minus 20 degrees Celsius until ready to perform protein quantification.Then transfer the sample to a new micro-centrifuge tube. Add one milliliter of fresh buffer solution and incubate until the next time point, as outlined in the text protocol. In this study, the conditions for fabricating PLGA electrospun fibers are examined.SEM analysis of blank, 0.05 nanomole, 0.5 nanomole, and 5 nanomole GRFT fibers indicates that GRFT modification has no effect on fiber morphology. All of the formations are seen to have similar average diameters, around 1.9 microns, demonstrating the consistency of the modified fabrication process across batches. The amount of GRFT conjugated in the electrospun fibers is then determined.The 5 nanomole modification contains 373 nanograms of GRFT, while the 0.5 and 0.05 modifications contains 165 and 42, respectively. This demonstrates that fibers conjugated with higher theoretical surface modification density result in more GRFT conjugated to the fiber. However, an inverse correlation can be seen in the resulting conjugation efficiency.Next, the amount of GRFT covalently conjugated to the fiber is assessed. Within the first four hours, 113 nanograms, 25 nanograms, and 10 nanograms of GRFT per milligram of electrospun fiber is detected in the 5 nanomole, 0.5 nanomole, and 0.05 nanomole theoretical modification concentrations, respectively. After four hours, negligible GRFT is detected in the release elute for all three formulations.This data indicates that the majority of GRFT is covalently bound to the electrospun fibers and that the surface absorbed GRFT is released within the first four hours. Once mastered, this technique can be done in approximately four days. Electrospinning the polymer fiber scaffold, requires two days to prepare the polymer solution and to electrospin.Whereas surface modification, subsequent drying and fiber characterization required at least two additional days. While attempting this procedure, it's important to remember to maintain the proper ratios of reagents to ensure optimal modification. Furthermore, it's also important to verify that the electospinning parameters are correct to ensure appropriate fiber morphology.After its development, this technique paved the way for researchers in the field of drug delivery to fabricate electrospun fibers, that not only encapsulate antiviral agents within the fiber, but have a capacity to conjugate active agents through the fiber surface. After watching this video, you should have a good understanding of how to electrospin polymeric fiber, perform surface modification of fiber with the anti-viral protein Griffithsin, and qualitative Griffithsin conjugated to the fiber. Don't forget that working with reagents such as HFIP, beta-mercaptoethanol, EDC and NHS can be extremely hazardous.Precautions such as proper application of PPE, and the use of chemical fume hoods, should always be taken while performing this procedure.

fabrication-characterization-griffithsin-modified-fiber-scaffolds-for

Research

JoVE Journal

Bioengineering

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Fabrication and Characterization of a Conformal Skin-like Electronic System for Quantitative, Cutaneous Wound Management

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of cutaneous wound healing on the skin. Existing methods, however, still rely on visual inspections through various microscopic tools and devices that normally include high-cost, sophisticated systems and require well trained personnel for operation and data analysis. Here, we describe methods and protocols to fabricate a conformal, skin-like electronics system that enables conformal lamination to the skin surface near the wound tissues, which provides recording of high fidelity electrical signals such as skin temperature and thermal conductivity. The methods of device fabrication provide details of step-by-step preparation of the microelectronic system that is completely enclosed with elastomeric silicone materials to offer electrical isolation. The experimental study presents multifunctional, biocompatible, waterproof, reusable, and flexible/stretchable characteristics of the device for clinical applications. Protocols of clinical testing provide an overview and sequential process of cleaning, testing setup, system operation, and data acquisition with the skin-like electronics, gently mounted on hypersensitive, cutaneous wound and contralateral tissues on patients.

This article presents methods to fabricate and characterize a conformal, skin-like electronic system and protocols for the use in clinical applications, particularly on cutaneous wound management.

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Interlinked Macroporous 3D Scaffolds from Microgel Rods

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions without further additives. Continuous photoinitiated on-chip gelation enables variation of the microgel aspect ratio, which determines the building block properties for the obtained constructs. Glycidyl methacrylate (GMA) or 2-aminoethyl methacrylate (AMA) monomers are copolymerized into the microgel network based on polyethylene glycol (PEG) star-polymers to achieve either epoxy or amine functionality. A focusing oil flow is introduced into the microfluidic outlet structure to ensure continuous collection of the functionalized microgel rods. Based on a recent publication, microgel rod-based constructs result in larger pores of several hundred micrometers and, at the same time, lead to overall higher scaffold stability in comparison to a spherical-based model. In this way, it is possible to produce higher-volume constructs with more free volume while reducing the amount of material required. The interlinked macroporous scaffolds can be picked up and transported without damage or disintegration. Amine and epoxy groups not involved in interlinking remain active and can be used independently for post-modification. This protocol describes an optimized method for the fabrication of microgel rods to form macroporous interlinked scaffolds that can be utilized for subsequent cell experiments.

Microgel rods with complementary reactive groups are produced via microfluidics with the ability to interlink in aqueous solution. The anisometric microgels jam and interlink into stable constructs with larger pores compared to spherical-based systems. Microgels modified with GRGDS-PC form macroporous 3D constructs that can be used for cell culture.

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions ...

Fabrication and Use of Dry Macroporous Alginate Scaffolds for Viral Transduction of T Cells

Genetic engineering of T cells for CAR-T cell therapy has come to the forefront of cancer treatment over the last few years. CAR-T cells are produced by viral gene transfer into T cells. The current gold standard of viral gene transfer involves spinoculation of retronectin-coated plates, which is expensive and time-consuming. There is a significant need for efficient and cost-effective methods to generate CAR-T cells. Described here is a method for fabricating inexpensive, dry macroporous alginate scaffolds, known as Drydux scaffolds, that efficiently promote viral transduction of activated T cells. The scaffolds are designed to be used in place of gold standard spinoculation of retronectin-coated plates seeded with virus and simplify the process for transducing cells. Alginate is cross-linked with calcium-D-gluconate and frozen overnight to create the scaffolds. The frozen scaffolds are freeze-dried in a lyophilizer for 72 h to complete the formation of the dry macroporous scaffolds. The scaffolds mediate viral gene transfer when virus and activated T cells are seeded together on top of the scaffold to produce genetically modified cells. The scaffolds produce &gt;85% primary T cell transduction, which is comparable to the transduction efficiency of spinoculation on retronectin-coated plates. These results demonstrate that dry macroporous alginate scaffolds serve as a cheaper and more convenient alternative to the conventional transduction method.

Herein is a protocol for creating dry macroporous alginate scaffolds that mediate efficient viral gene transfer for use in genetic engineering of T cells, including T cells for CAR-T cell therapy. The scaffolds were shown to transduce activated primary T cells with &gt;85% transduction.

Genetic engineering of T cells for CAR-T cell therapy has come to the forefront of cancer treatment over the last few years. CAR-T cells are produced by ...