Authors are grateful to C. Luzuriaga for the support in the porosimetry measurements. Authors also thanks to Ministerio de Econom&#237;a y Competitividad of Spain for financial support (Project MAT2014-59116-C2-2-R) and PIUNA (ref. 2018-15). The authors also would like to acknowledge Dr. Amir Maldonado from Departamento de F&#237;sica-UNISON for support and helpful comments and Dr. SE Burruel-Ibarra from DIPM-UNISON for SEM images and Rubio Pharma y Asociados S. A. de C. V. for financial support. ME Mart&#237;nez-Barbosa would like to thank CONACyT (M&#233;xico) projects No. 104931 and No. 256753, besides the financial support from Red Tem&#225;tica de Nanociencias y Nanotecnolog&#237;a del programa de Redes Tem&#225;ticas del CONACyT. And, also project USO316001081. MD Figueroa-Pizano would like to acknowledge CONACyT for financial support (scholarship 373321).

1. Preparation of chitosan-PVA hydrogels
<ol>
	<li>Prepare 2% (w/w) chitosan and 10% (w/w) PVA solutions. Dissolve 0.2 g of chitosan in 10 mL of 0.1 M CH3COOH solution (previously filtered) at room temperature and maintain continuous mechanical stirring overnight. Dissolve 1 g of PVA in 10 mL of distilled water and stir at 80 &#176;C for 1 h.</li>
	<li>Mix both solutions 1:1 using a magnetic stirrer until they are homogeneous at room temperature, and pour the mixtures on Petri dishes. Leave the samples for 2 h at atmospheric pressure to degas.</li>
	<li>Freeze the hydrogels at -4 &#176;C, -20 &#176;C or -80 &#176;C for 20 h and 4 cycles (samples CP4-4, CP4-20 and CP4-80, respectively). Freeze another hydrogel at -80 &#176;C for 20 h using 5 or 6 freezing cycles (samples CP5-80 and CP6-80). After the third freezing cycle, wash the hydrogels with deionized water. At the end, freeze-dry the hydrogels at -46 &#176;C for 48 h and store for further characterization (methodology adapted from2).</li>
</ol>
2. FT-IR characterization
<ol>
	<li>Place a little piece (1 mm x 2 mm) of hydrogel in the FT-IR spectrometer in ATR mode. Take the FT-IR spectra from 4000 to 600 cm-1 (2 cm-1 of resolution and average of 32 scans).</li>
</ol>
3. Swelling assays
<ol>
	<li>Cut out discs (13 mm in diameter and 10 mm in height) from the hydrogel and weigh them. Incubate the discs in 50 mL of deionized water with shaking at 25 &#176;C. Repeat three times.</li>
	<li>Every 30 min remove the sample from the medium, blotter to eliminate the excess of water, and weigh. Calculate the swelling degree using the equation 1 and calculate the equilibrium state of swelling, <img alt="Equation 1" src="/files/ftp_upload/59636/59636eq1.jpg" />, at 24 h using the equation 2. 
	<img alt="Equation 2" src="/files/ftp_upload/59636/59636eq2.jpg" />) 
	Where <img alt="Equation 3" src="/files/ftp_upload/59636/59636eq3.jpg" /> is the weight of the dry hydrogel and <img alt="Equation 4" src="/files/ftp_upload/59636/59636eq4.jpg" /> is the weight of the wet hydrogel. 
	<img alt="Equation 5" src="/files/ftp_upload/59636/59636eq5.jpg" /></li>
</ol>
4. Electronic Microscopy
<ol>
	<li>Cover a little piece of hydrogel with a thin gold layer (30 s and 10 mA) in a sputter coater.</li>
	<li>Put the sample in a scanning electron microscope (SEM). Analyze the samples under vacuum at 20 kV and take the images with a 500x and 1500x magnification.</li>
</ol>
5. Porosimetry
<ol>
	<li>Place discs 15 mm in diameter weighing around 0.26 g into the penetrometer (a solid penetrometer, having a bulk volume of 0.3660 mL and 5.7831 mL of stem volume). Analyze the porosity and pore size by Mercury Intrusion Porosimetry (MIP).</li>
	<li>Conduct the experiment in the hysteresis mode (intrusion-extrusion). Measure the total intrusion volume (mL/g), total pore area (m2/g), pore diameter (&#181;m), porosity (%), permeability (mDarcy) and tortuosity. Repeat twice.</li>
</ol>
6. Drug loading and release
<ol>
	<li>Before loading, prepare 4 L of 15 mg/L diflunisal solution and stir overnight. Confirm the concentration of the solution by UV-Vis spectroscopy (initial concentration). Indeed, swell 400 mg of freeze-dried samples of hydrogel in 6 mL of distilled water for 24 h.</li>
	<li>For loading, fill a flask with 50 mL of diflunisal solution and maintain at 25 &#176;C with constant stirring. Submerge each swelled hydrogel in the flask.
	<ol>
		<li>Take aliquots of remaining diflunisal solution (2 mL) at different times in order to determine the plateau region of the curve, for example: 3, 6, 24, 27, 30 and 48 h. After 24 h replace the solution with a fresh one.</li>
	</ol>
	</li>
	<li>Measure the absorbance at 252 nm of each aliquot, and determine the concentration of diflunisal present in the solution, using a calibration curve of diflunisal. Calculate the amount of diflunisal retained in the hydrogel at 24 and 48 h, as the difference of initial and final concentrations, taking into account the total volume (56 mL).
	<ol>
		<li>Determine the encapsulation efficiency (EE) using the equation 3. 
		<img alt="Equation 6" src="/files/ftp_upload/59636/59636eq6.jpg" /></li>
		<li>Freeze the loaded hydrogels at -80 &#176;C and lyophilize them at -50 &#176;C.</li>
	</ol>
	</li>
	<li>For drug release, submerge 300 mg of freeze-dried diflunisal loaded hydrogels in 50 mL of phosphate buffer (pH 7.4) at 25 &#176;C. Maintain constant stirring. Withdraw aliquots of 2 mL at different times and replace with fresh medium to keep a constant volume.
	<ol>
		<li>Determinate the diflunisal released spectrophotometrically at 252 nm, according to a calibration curve.</li>
	</ol>
	</li>
	<li>Deduce the predominant drug release mechanism in the hydrogels adjusting the drug release data corresponding to the first 60%, to the Korsmeyer-Peppas model (Equation 4), to obtain the kinetic (k) and the diffusion (n) constants. The n values indicate the mechanism of drug release24,25. Then, n values close to 0.5 are related to Fickian diffusion, meanwhile values of 0.5-1.0 for anomalous transport, where are involved diffusion and relaxation chains, and finally, values of 1.0 are related to case II transport. 
	<img alt="Equation 7" src="/files/ftp_upload/59636/59636eq7.jpg" />
	<ol>
		<li>To confirm the results, use the Higuchi, First order, and Zero order mathematical models (Equations 5 to 7) and select the better fit.</li>
		<li><img alt="Equation 8" src="/files/ftp_upload/59636/59636eq8.jpg" /> 
		<img alt="Equation 9" src="/files/ftp_upload/59636/59636eq9.jpg" /> 
		<img alt="Equation 10" src="/files/ftp_upload/59636/59636eq10.jpg" /> 
		where t represents the release time, Mt the amount of drug delivered at a given time, and M&#8734; the total amount of drug delivered at the end of the process.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

The freeze-thawing method is a suitable process to prepare biocompatible hydrogels focused in biomedical, pharmaceutical or cosmetical applications34,35,36. The most important advantage of this method, compared with other well-known methods to prepare hydrogels, is that crosslinking agent use is avoided, which could cause an inflammatory response or adverse effects in the human body34. This is a versatile...

Recently, hydrogels have attracted great interest in the biomedical field because they are three-dimensional networks with high water content and are soft and flexible, so they can mimic natural tissues easily1. Also, they do not dissolve in aqueous medium at physiological temperature and pH but present a large swelling2. Hydrogels can act as tissue engineering scaffolds, hygiene products, contact lenses, and wound dressings; because they can trap and release active compounds and drugs, they are used as drug delivery systems3. Depending on their application, hydrogels can be made from natural or synthetic polymers, or a combination of both, in order to obtain the best characteristics4.
The properties of hydrogels are a consequence of many physical and chemical factors. At the physical level, their structure and morphology depend on their porosity, pore size and pore distribution5. At the chemical and molecular level, the polymer type, the hydrophilic group content in the polymer chain, the crosslinking point type, and the cross-linking density are the factors that determine the swelling capacity and the mechanical properties6,7.
According to the type of crosslinking agent used to form the network, the hydrogels are classified as chemical hydrogels or physical hydrogels. Chemical hydrogels are joined by covalent interactions between their chains, which are formed through UV and gamma irradiation or using a crosslinking agent7,8. Chemical hydrogels usually are strong and resistant but, generally, the crosslinking agent is toxic to the cells and its removal is difficult, so its application is limited. On the other hand, physical hydrogels form by the connection of the polymer chains through non-covalent interactions, avoiding the use of crosslinking agents4,9. The main non-covalent interactions in the network are hydrophobic interactions, electrostatic forces, complementary and hydrogen bounds7.
Poly(vinyl alcohol) (PVA, Figure 1a) is a synthetic and water-soluble polymer with excellent mechanical performance and biocompatibility that can from crosslink agent-free hydrogels through the freeze-thawing method10,11. This polymer has the capacity to form concentrated zones of hydrogen bonds between -OH groups of their chains (crystalline zones) when they are freezing12. These crystalline zones act as crosslinking points in the network, and they are promoted by two events: the approaching of the polymer chains when the crystal water expands and the PVA conformational changes from isotactic to syndiotactic PVA during freeze13. Because of the freeze-drying, the water crystals are sublimated, leaving void spaces that are the pores in the hydrogel14. To obtain hydrogels with better properties, PVA can been easily combined with other polymers.
In that sense, chitosan constitutes an option as it is the only biopolymer from natural sources with positive charges. It is obtained by the deacetylation of chitin and it is composed of random combinations of &#946;-1,4 linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit)15,16 (Figure 1b). Chitosan is biodegradable by human enzymes and it is biocompatible. Also, by its cationic nature, it can interact with the negative charge of the cell surface, and this property has been associated with its antimicrobial activity17. This polymer is easy to process; however, their mechanical properties are not sufficient and some materials have been added to form complexes with better characteristics.
Considering specific characteristics of chitosan and PVA, the successful manufacture of hydrogels has been reached by the freeze-thawing method2,18 to avoid the use of toxic crosslinking agents. In chitosan-PVA hydrogels, the crystalline zones of PVA are also formed, and chitosan chains are interpenetrated and form simple hydrogen bonds with -NH2 groups and -OH groups in PVA. The final chitosan-PVA hydrogel is mechanically stable, with high rates of swelling and low toxicity, and with antibacterial effect18. However, depending on the freezing conditions used in the preparation (temperature, time and number of cycles), the final characteristics may change. Some studies report that increasing the number of freezing cycles decreases the swelling degree and increases the tensile strength19,20. In order to strengthen the network, other agents such as gamma and UV radiation and chemical crosslinkers have been used additionally after the freeze-thawed preparation21,22,23. Hydrogels with a higher chitosan proportion have a more porous network and high swelling capacity but less strength and thermal stability. In this context, it is important to consider the preparation conditions to obtain suitable hydrogels for their target application.
The purpose of this work is to present in detail how the freezing conditions (temperature of freezing and number of cycles) affect the final characteristics of CS-PVA hydrogels. FT-IR spectra, morphological and porosity characteristics and swelling capacity were evaluated, as well as drug loading and release capacity. In the release studies, diflunisal (Figure 1c) was used as model drug, due to its size suitable to the hydrogel structure.

<table><tbody><tr><td>&lt;strong&gt;Materials:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Chitosan medium molecular weight</td><td>Sigma-Aldrich</td><td>448877</td><td>Mw determined by capillary viscometry (637,000 Da) and deacetylation degree of 70%</td></tr><tr><td>Diflunisal (2&#039;-4&#039;-difluoro-4-hydroxy-3-biphenyl-carboxylicacid)</td><td>Merck</td><td></td><td></td></tr><tr><td>Glacial acetic acid</td><td>Sigma-Aldrich</td><td>1005706</td><td></td></tr><tr><td>Poly(vinyl alcohol)</td><td>Sigma-Aldrich</td><td>341584</td><td>Mw 89,000-98,000, 99+% hydrolyzed</td></tr><tr><td>&lt;strong&gt;Equipment:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cressington Sputter Coater 108 auto</td><td>TED PELLA INC</td><td></td><td></td></tr><tr><td>Cryodos Lyophilizator</td><td>Telstar</td><td></td><td></td></tr><tr><td>Falcon tubes</td><td>Thermo Fisher Company</td><td></td><td></td></tr><tr><td>FT-IR spectroscopy</td><td>Nicolet iS50</td><td>in ATR mode</td><td></td></tr><tr><td>Lyophilizator</td><td>LABCONCO</td><td></td><td></td></tr><tr><td>Micromeritics Autopore IV 9500</td><td>Micromeritics</td><td></td><td></td></tr><tr><td>Scanning electron microscope</td><td>Pemtron SS-300LV</td><td></td><td></td></tr><tr><td>UV-visible spectrophotometer</td><td>Agilent 8453</td><td></td><td></td></tr></tbody></table>

a freeze-thawing method to prepare chitosan-poly(vinyl alcohol) hydrogels without crosslinking agents and diflunisal release studies

Chitosan-poly(vinyl alcohol) hydrogels can be produced by the freeze-thawing method without using toxic crosslinking agents. The applications of these systems are limited by their characteristics (e.g., porosity, flexibility, swelling capacity, drug loading and drug release capacity), which depend on the freezing conditions and the kind and ratio of polymers. This protocol describes how to prepare hydrogels from chitosan and poly(vinyl alcohol) at 50/50 w/w % of polymer composition and varying the freezing temperature (-4 &#176;C, -20 &#176;C, -80 &#176;C) and freeze-thawing cycles (4, 5, 6 freezing cycles). FT-IR spectra, SEM micrograph and porosimetry data of hydrogels were obtained. Also, the swelling capacity and drug loading and release of diflunisal were assessed. Results from SEM micrographs and porosimetry show that the pore size decreases, while the porosity increases at lower temperatures. The swelling percentage was higher at the minor freezing temperature. The release of diflunisal from the hydrogels has been studied. All the networks maintain the drug release for 30 h and it has been observed that a simple diffusion mechanism regulates the diflunisal release according to Korsmeyer-Peppas and Higuchi models.

Hydrogels preparation 
Chitosan-PVA hydrogels were obtained at -4 &#176;C, -20 &#176;C and -80 &#176;C with 4 freezing cycles and at -80 &#176;C with 5 and 6 freezing cycles by the previously reported freeze-thawing method2. All hydrogels were homogeneous, semi-transparent, flexible and resistant against manipulation.
FT-IR characterization 
The FT-IR spectra ar...

Watch this Scientific Journal Video about A Freeze-Thawing Method to Prepare Chitosan-Polyvinyl alcohol Hydrogels Without Crosslinking Agents and Diflunisal Release Studies at JoVE.com

A Freeze-Thawing Method to Prepare Chitosan-Polyvinyl alcohol Hydrogels Without Crosslinking Agents and Diflunisal Release Studies

The freezing-thawing method is used to produce chitosan-poly(vinyl alcohol) hydrogels without crosslinking agents. For this method, it is important to consider the freezing conditions (temperature, number of cycles) and polymer ratio, which can affect the properties and applications of the obtained hydrogels.

A Freeze-Thawing Method to Prepare Chitosan-Poly(vinyl alcohol) Hydrogels Without Crosslinking Agents and Diflunisal Release Studies

Chitosan-poly(vinyl alcohol) hydrogels can be produced by the freeze-thawing method without using toxic crosslinking agents. The applications of these ...

a-freeze-thawing-method-to-prepare-chitosan-poly-vinyl-alcohol

Research

JoVE Journal

Bioengineering

17.4K Views.  University of Sonora. Dit protocol is belangrijk omdat onze vriesdooimethode een geschikt proces is om biocompatibele hydrogels voor te bereiden voor gebruik in biomedische, farmaceutische of cosmetische toepassingen. Het belangrijkste voordeel van deze methode is dat het geen gebruik maakt van chemische crosslinking middelen die een aangeboren afwijking kan veroorzaken. Ook de bevriezing voorwaarde gebruikt in deze methode controle van de uiteindelijke eigenschap van hydrogels zoals graden.Deze methode kan...

Video: A Freeze-Thawing Method to Prepare Chitosan-Polyvinyl alcohol Hydrogels Without Crosslinking Agents and Diflunisal Release Studies

Preparation and Characterization of SDF-1&#945;-Chitosan-Dextran Sulfate Nanoparticles

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under appropriate conditions. The glycan nanoparticles are useful carriers for protein factors, which facilitate the in vivo delivery of the proteins and sustain their retention in the targeted tissue. The glycan polyelectrolyte complexes are also ideal for protein delivery, as the incorporation is carried out in aqueous solution, which reduces the likelihood of inactivation of the proteins. Proteins with a heparin-binding site adhere to dextran sulfate readily, and are, in turn, stabilized by the binding. These particles are also less inflammatory and toxic when delivered in vivo. In the protocol described below, SDF-1&#945; (Stromal cell-derived factor-1&#945;), a stem cell homing factor, is first mixed and incubated with dextran sulfate. Chitosan is added to the mixture to form polyelectrolyte complexes, followed by zinc sulfate to stabilize the complexes with zinc bridges. The resultant SDF-1&#945;-DS-CS particles are measured for size (diameter) and surface charge (zeta potential). The amount of the incorporated SDF-1&#945; is determined, followed by measurements of its in vitro release rate and its chemotactic activity in a particle-bound form.

The objective of this protocol is to incorporate SDF-1&#945;, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles. The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1&#945; from the nanoparticles.

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under ...

Chemistry

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Biochemistry

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. Compared to other analytical separation techniques, such as chromatography, CE is cheap, robust and effectively requires no sample preparation (for a number of complex natural matrices or polymeric samples). CE is fast and can be used to follow the evolution of mixtures in real time (e.g., chemical reaction kinetics), as the signals observed for the separated compounds are directly proportional to their quantity in solution.
Here, the efficiency of CE is demonstrated for monitoring the covalent grafting of peptides onto chitosan films for subsequent biomedical applications. Chitosan&#39;s antimicrobial and biocompatible properties make it an attractive material for biomedical applications such as cell growth substrates. Covalently grafting the peptide RGDS (arginine - glycine - aspartic acid - serine) onto the surface of chitosan films aims at improving cell attachment. Historically, chromatography and amino acid analysis have been used to provide a direct measurement of the amount of grafted peptide. However, the fast separation and absence of sample preparation provided by CE enables equally accurate yet real-time monitoring of the peptide grafting process. CE is able to separate and quantify the different components of the reaction mixture: the (non-grafted) peptide and the chemical coupling agents. In this way the use of CE results in improved films for downstream applications.
The chitosan films were characterized through solid-state NMR (nuclear magnetic resonance) spectroscopy. This technique is more time-consuming and cannot be applied in real time, but yields a direct measurement of the peptide and thus validates the CE technique.

Free solution capillary electrophoresis is a fast, cheap and robust analytical method that enables the quantitative monitoring of chemical reactions in real time. Its utility for rapid, convenient and precise analysis is demonstrated here through analysis of covalent peptide grafting onto chitosan films for improved cell adhesion.

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. ...

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is prepared by mixing solutions of glycol chitosan with a synthesized benzaldehyde terminated polymer gelator, and hydrogels are efficiently obtained in several minutes at room temperature. By varying ratios between glycol chitosan, polymer gelator, and water contents, versatile hydrogels with different gelation times and stiffness are obtained. When damaged, the hydrogel can recover its appearances and modulus, due to the reversibility of the dynamic imine bonds as crosslinkages. This self-healable property enables the hydrogel to be injectable since it can be self-healed from squeezed pieces to an integral bulk hydrogel after the injection process. The hydrogel is also multi-responsive to many bio-active stimuli due to different equilibration statuses of the dynamic imine bonds. This hydrogel was confirmed as bio-compatible, and L929 mouse fibroblast cells were embedded following standard procedures and the cell proliferation was easily assessed by a 3D cell cultivation process. The hydrogel can offer an adjustable platform for different research where a physiological mimic of a 3D environment for cells is profited. Along with its multi-responsive, self-healable, and injectable properties, the hydrogels can potentially be applied as multiple carriers for drugs and cells in future bio-medical applications.

Here we describe a facile preparation of chitosan-based injectable hydrogels using dynamic imine chemistry. Methods to adjust the hydrogel&#8217;s mechanical strength and its application in 3D cell culture are presented.

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is ...

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

In this article, we describe a protocol for fabricating an amelogenin-chitosan hydrogel for superficial enamel reconstruction. Organized in situ growth of apatite crystals in the hydrogel formed a dense enamel-restoration interface, which will improve the effectiveness and durability of restorations.

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.

Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial ...

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), their ultimate clinical use has been hampered by the lack of biologically-relevant degradation observed for most smart materials. This is particularly true for temperature-responsive hydrogels, which are almost uniformly based on polymers that are functionally non-degradable (e.g., poly(N-isopropylacrylamide) (PNIPAM) or poly(oligoethylene glycol methacrylate) (POEGMA)). As such, to effectively translate the potential of thermoresponsive hydrogels to the challenges of remote-controlled or metabolism-regulated drug delivery, cell scaffolds with tunable cell-material interactions, theranostic materials with the potential for both imaging and drug delivery, and other such applications, a method is required to render the hydrogels (if not fully degradable) at least capable of renal clearance following the required lifetime of the material. To that end, this protocol describes the preparation of hydrolytically-degradable hydrazone-crosslinked hydrogels on multiple length scales based on the reaction between hydrazide and aldehyde-functionalized PNIPAM or POEGMA oligomers with molecular weights below the renal filtration limit. Specifically, methods to fabricate degradable thermoresponsive bulk hydrogels (using a double barrel syringe technique), hydrogel particles (on both the microscale through the use of a microfluidics platform facilitating simultaneous mixing and emulsification of the precursor polymers and the nanoscale through the use of a thermally-driven self-assembly and cross-linking method), and hydrogel nanofibers (using a reactive electrospinning strategy) are described. In each case, hydrogels with temperature-responsive properties similar to those achieved via conventional free radical cross-linking processes can be achieved, but the hydrazone cross-linked network can be degraded over time to re-form the oligomeric precursor polymers and enable clearance. As such, we anticipate these methods (which may be generically applied to any synthetic water-soluble polymer, not just smart materials) will enable easier translation of synthetic smart materials to clinical applications.

Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), ...

Preparation of Expanded Chitin Foams and their Use in the Removal of Aqueous Copper

Chitin is an underexploited, naturally abundant, mechanically robust, and chemically resistant biopolymer. These qualities are desirable in an adsorbent, but chitin lacks the necessary specific surface area, and its modification involves specialized techniques and equipment. Herein is described a novel chemical procedure for expanding chitin flakes, derived from shrimp shell waste, into foams with higher surface area. The process relies on the evolution of H2 gas from the reaction of water with NaH trapped in a chitin gel. The preparation method requires no specialized equipment. Powder X-ray diffraction and N2-physisorption indicate that the crystallite size decreases from 6.6 nm to 4.4 nm and the specific surface area increases from 12.6 &#177; 2.1 m2/g to 73.9 &#177; 0.2 m2/g. However, infrared spectroscopy and thermogravimetric analysis indicate that the process does not change the chemical identity of the chitin. The specific Cu adsorption capacity of the expanded chitin increases in proportion to specific surface area from 13.8 &#177; 2.9 mg/g to 73.1 &#177; 2.0 mg/g. However, the Cu adsorption capacity as a surface density remains relatively constant at an average of 10.1 &#177; 0.8 atom/nm2, which again suggests no change in the chemical identity of the chitin. This method offers the means to transform chitin into a higher surface area material without sacrificing its desirable properties. Although the chitin foam is described here as an adsorbent, it can be envisioned as a catalyst support, thermal insulator, and structural material.

This study describes a method to expand chitin into a foam by chemical techniques that require no specialized equipment.

Chitin is an underexploited, naturally abundant, mechanically robust, and chemically resistant biopolymer. These qualities are desirable in an adsorbent, ...

A Freeze-Thawing Method to Prepare Chitosan-Poly(vinyl alcohol) Hydrogels Without Crosslinking Agents and Diflunisal Release Studies

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