The C57BL/6 mice were purchased from Zhejiang University School of Medicine Sir Run Run Shaw Hospital. The New Zealand rabbits were purchased from Zhejiang University. The animals were maintained in natural light-dark cycle conditions and given food and drinking water freely. All experimental procedures were approved ethically by the institutional guidelines of the Zhejiang University Ethics Committee standard guidelines (ZJU20200156) and Zhejiang University School of Medicine Sir Run Run Shaw Hospital Animal Care and Use Committee, which conformed to the NIH Guide for the Care and Use of Laboratory Animals (SRRSH202107106).
1. Synthesis of NB-COOH
<ol>
	<li>Prepare 4-hydroxy-3-(methoxy-D3) benzaldehyde (8.90 g, 58.5 mM, 1.06 equivalents [eq.]), potassium carbonate (10.2 g, 73.8 mM, 1.34 eq.), and methyl 4-bromobutyrate (9.89 g, 55.0 mM, 1.0 eq.) based on the protocol proposed in the previous study10. Dissolve the compounds in 40 mL of N, N-dimethylformamide (DMF) and stir at ambient temperature for 16 h.</li>
	<li>Add 200 mL of 0 &#176;C water to the mixture and precipitate the mixture to obtain a crude product.</li>
	<li>Repeatedly dissolve the crude product in DMF and then precipitate for five cycles. Precipitate the crude product and dry it at 80 &#176;C for 2 h to obtain the early product.</li>
</ol>
2. Chemical modification and processing
<ol>
	<li>Perform the ipso substitution of methyl 4-(4-formyl-2-methoxyphenoxy methoxyphenyl) butanoic acid methyl ester as described below.</li>
	<li>Add 9.4 g of methyl 4-(4-formyl-2-methoxyphenoxy) butanoate (37.3 mM, 1 eq.) slowly to a precooled (-2 &#176;C) solution of 70% nitric acid (140 mL) and stir at -2 &#176;C for 3 h. 
	NOTE: Depending on the temperature of the nitration reaction, the ipso substitution of the formyl moiety will occur.</li>
	<li>Filter the mixture (~9.0 g) with 200 mL of 0 &#176;C water, then purify it in DMF to precipitate a solid product.</li>
	<li>Hydrolyze the solid product in trifluoroacetic acid (TFA)/H2O, 1:10 v/v (100 mL) at 90 &#176;C and dry. Remove the solvent under 80 kPa to obtain the final intermediate product, a dry pale-yellow powder.</li>
	<li>Dissolve the intermediate product (7.4 g, 23.8 mM, 1.0 eq.) in tetrahydrofuran (THF)/ethanol, 1:1 v/v (100 mL). Then add 1.43 g of NaBH4 (35.7 mM, 1.5 eq.) slowly at 0 &#176;C. After 3 h, remove all solvents under a vacuum and suspend the residue in a 1:1 water and dichloromethane solution (50 mL each).</li>
	<li>Prepare dichloromethane to extract the product from the aqueous layer. Remove the organic layer and dry over magnesium sulfate.</li>
	<li>Purify the crude product by silica gel column chromatography using DCM/MeOH at a 10:1 ratio (1% TEA). Finally, obtain 5.31 g (18.6 mM, 78.3%) of relatively pure yellowish powder NB-COOH.</li>
</ol>
3. Synthesis of gelatin-NB
<ol>
	<li>Prepare 5 g of gelatin for one batch of modification. Prepare a homogeneous gelatin solution by dissolving 5 g of gelatin in 100 mL of deionized water and store at 37 &#176;C. 
	NOTE: Here, the original 33 x 10-5 moles &#949;-amino groups/g gelatin11 is defined.</li>
	<li>Define the feed ratio (FR) as the molar ratio between NB groups and primary amino groups in gelatin. In this study, 53 mg of NB with 1 g of gelatin was defined as FRNB = 1.</li>
	<li>Dissolve 1,060 mg of NB-COOH in 5 mL of dimethyl sulfoxide (DMSO) to activate the carboxyl groups of NB-COOH. Since the NB-group is sensitive to ultraviolet (UV) light when in solution, always keep it away from light.</li>
	<li>Add 746 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodimide hydrochloride (EDC) into the NB-COOH DMSO solution and stir for 5 min. After EDC has dissolved, add 448 mg of N-hydroxysuccinimide (NHS) and stir for 5 min.</li>
	<li>Use a dropping funnel to slowly drop the mixture at a rate of 0.5 mL/min into the dissolved gelatin solution with vigorous stirring to react at 45 &#176;C for 4 h.</li>
</ol>
4. Purification and storage of the product
<ol>
	<li>Dialyze the gelatin-NB solution against excess deionized water for at least 3 days, then collect, freeze, and lyophilize it to obtain the gelatin-NB foams. Keep the foams in a desiccator in the dark for further use.</li>
	<li>Dissolve the freeze-dried gelatin-NB foams in deionized water at 37 &#176;C, immediately before use.</li>
</ol>

<ol>
	<li>Tam, R. Y., Smith, L. J., Shoichet, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+cellular+microenvironments+with+photo-+and+enzymatically+responsive+hydrogels:+toward+biomimetic+3D+cell+culture+models.">Engineering cellular microenvironments with photo- and enzymatically responsive hydrogels: toward biomimetic 3D cell culture models.</a> Accounts of Chemical Research. 50 (4), 703-713 (2017).</li><li>Xu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioadhesive+hydrogels+demonstrating+pH-independent+and+ultrafast+gelation+promote+gastric+ulcer+healing+in+pigs.">Bioadhesive hydrogels demonstrating pH-independent and ultrafast gelation promote gastric ulcer healing in pigs.</a> Science Translational Medicine. 12 (558), (2020).</li><li>Zheng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+self-assembly+of+herbal+small+molecules+into+sustained+release+hydrogels+for+treating+neural+inflammation.">Directed self-assembly of herbal small molecules into sustained release hydrogels for treating neural inflammation.</a> Nature Communications. 10 (1), 1604 (2019).</li><li>Seif-Naraghi, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+efficacy+of+an+injectable+extracellular+matrix+hydrogel+for+treating+myocardial+infarction.">Safety and efficacy of an injectable extracellular matrix hydrogel for treating myocardial infarction.</a> Science Translational Medicine. 5 (173), (2013).</li><li>Mao, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GelNB+molecular+coating+as+a+biophysical+barrier+to+isolate+intestinal+irritating+metabolites+and+regulate+intestinal+microbial+homeostasis+in+the+treatment+of+inflammatory+bowel+disease.">GelNB molecular coating as a biophysical barrier to isolate intestinal irritating metabolites and regulate intestinal microbial homeostasis in the treatment of inflammatory bowel disease.</a> Bioactive Materials. 19, 251-267 (2022).</li><li>Nan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+elastic+and+fatigue-resistant+natural+protein-reinforced+hydrogel+electrolyte+for+reversible-compressible+quasi-solid-state+supercapacitors.">A highly elastic and fatigue-resistant natural protein-reinforced hydrogel electrolyte for reversible-compressible quasi-solid-state supercapacitors.</a> Advanced Science. 7 (14), 2000587 (2020).</li><li>Matsumoto, K., Sakikawa, N., Miyata, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermo-responsive+gels+that+absorb+moisture+and+ooze+water.">Thermo-responsive gels that absorb moisture and ooze water.</a> Nature Communications. 9 (1), 2315 (2018).</li><li>Liu, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=resilient,+adhesive,+and+anti-freezing+hydrogels+cross-linked+with+a+macromolecular+cross-linker+for+wearable+strain+sensors.">resilient, adhesive, and anti-freezing hydrogels cross-linked with a macromolecular cross-linker for wearable strain sensors.</a> ACS Applied Materials &amp; Interfaces. 13 (35), 42052-42062 (2021).</li><li>Hong, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+strongly+adhesive+hemostatic+hydrogel+for+the+repair+of+arterial+and+heart+bleeds.">A strongly adhesive hemostatic hydrogel for the repair of arterial and heart bleeds.</a> Nature Communications. 10 (1), 2060 (2019).</li><li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-integratable+and+biocompatible+photogelation+by+the+imine+crosslinking+reaction.">Tissue-integratable and biocompatible photogelation by the imine crosslinking reaction.</a> Advanced Materials. 28 (14), 2724-2730 (2016).</li><li>Ofner, C. M., Bubnis, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+and+swelling+evaluations+of+amino+group+crosslinking+in+gelatin+and+modified+gelatin+matrices.">Chemical and swelling evaluations of amino group crosslinking in gelatin and modified gelatin matrices.</a> Pharmaceutical Research. 13 (12), 1821-1827 (1996).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+long-term+retaining+molecular+coating+for+corneal+regeneration.">A long-term retaining molecular coating for corneal regeneration.</a> Bioactive Materials. 6 (12), 4447-4454 (2021).</li><li>Liang, Y., Li, Z., Huang, Y., Yu, R., Guo, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-dynamic-bond+cross-linked+antibacterial+adhesive+hydrogel+sealants+with+on-demand+removability+for+post-wound-closure+and+infected+wound+healing.">Dual-dynamic-bond cross-linked antibacterial adhesive hydrogel sealants with on-demand removability for post-wound-closure and infected wound healing.</a> ACS Nano. 15 (4), 7078-7093 (2021).</li></ol>

The authors have nothing to disclose.

Adhesive materials are a new class of material. More and more researchers are committed to the synthesis of various types of adhesive materials, and are trying to find their applications in biotechnology, tissue engineering, regenerative medicine, and other fields, which has led to vigorous development in recent years. In addition to focusing on the strong adhesion of adhesive materials, researchers are also paying more attention to other properties, such as injectability, self-healing, hemostatic, antibacterial, control...

Hydrogel is a type of three-dimensional polymer formed by water swelling. In particular, hydrogel derived from an extracellular matrix is widely used in the field of biosynthesis and regenerative medicine because of its excellent biocompatibility and therapeutic effectiveness1. Hydrogels have been reported for the treatment of gastric ulcers, neuritis, myocardial infarction2,3,4, and other diseases. Further, it has been proved that gelatin-NB can promote the outcome of inflammation ininflammatory bowel disease (IBD)5. Traditional hydrogels include gellan gum, gelatin, hyaluronic acid, polyethylene glycol (PEG), layered, hydrophobic/hydrophilic, alginate/polyacrylamide, double network, and polyamphoteric hydrogels6, all of which have good histocompatibility and mechanical properties. However, these traditional hydrogels are vulnerable to moisture and air in the environment. If they are exposed to air for a long time, they will lose water and dry; if they are immersed in the water for a long time, they will absorb water and expand7, thus reducing their flexibility and mechanical function. In addition, maintaining the tissue adhesion of conventional hydrogels is a major challenge8.
Based on this, we designed and synthesized a nanoscale hydrogel gelatin-NB, which is a novel hydrogel formed by modifying biological gelatin with NB (Figure 1). NB has a strong adhesion ability to -NH2 on the tissue, which can form a large number of C = N bonds, thus increasing the adhesiveness of the hydrogel-tissue interface. This strong adhesion can make the hydrogel firmly adhere to the tissue surface, thus forming a nano-level molecular coating. In the team&#39;s previous studies, it has been confirmed that this kind of modified hydrogel coating has improved tissue adhesion9; it can stably adhere to corneal and intestinal organs and tissues and play anti-inflammation, barrier isolation, and regeneration promotion roles. The goal is to introduce the specific synthesis process of gelatin-NB in detail here, so that gelatin-NB can be applied in more scenarios of damage repair. Moreover, we encourage other researchers to further strengthen and expand the nature of this material to suit more application scenarios.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-(3Dimethylaminopropyl)-3-ethylcarbodimide hydrochloride (EDC)</td>
			<td>Aladdin</td>
			<td>L287553</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Hydroxy-3-(methoxy-D3) benzaldehyde</td>
			<td>Shanghai Acmec Biochemical Co., Ltd</td>
			<td>H946072</td>
			<td></td>
		</tr>
		<tr>
			<td>DCM</td>
			<td>Aladdin</td>
			<td>D154840</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Sigma-Aldrich</td>
			<td>270997</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>20-139</td>
			<td></td>
		</tr>
		<tr>
			<td>dimethylformamide (DMF)</td>
			<td>Sigma-Aldrich</td>
			<td>PHR1553</td>
			<td></td>
		</tr>
		<tr>
			<td>gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>1288485</td>
			<td></td>
		</tr>
		<tr>
			<td>magnesium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>MeOH</td>
			<td>Sigma-Aldrich</td>
			<td>1424109</td>
			<td></td>
		</tr>
		<tr>
			<td>methyl 4-(4-formyl-2-methoxyphenoxy methoxyphenyl) butanoic acid methyl ester</td>
			<td>chemsrc</td>
			<td>141333-27-9</td>
			<td></td>
		</tr>
		<tr>
			<td>methyl 4-bromobutyrate</td>
			<td>Aladdin</td>
			<td>M158832</td>
			<td></td>
		</tr>
		<tr>
			<td>NaBH4</td>
			<td>Sigma-Aldrich</td>
			<td>215511</td>
			<td></td>
		</tr>
		<tr>
			<td>N-hydroxysuccinimide (NHS)</td>
			<td>Aladdin</td>
			<td>D342712</td>
			<td></td>
		</tr>
		<tr>
			<td>nitric acid</td>
			<td>Sigma-Aldrich</td>
			<td>225711</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>209619</td>
			<td></td>
		</tr>
		<tr>
			<td>SEM (Nova Nano 450)</td>
			<td>Thermo FEI</td>
			<td>17024560</td>
			<td></td>
		</tr>
		<tr>
			<td>THF/EtOH</td>
			<td>Aladdin</td>
			<td>D380010</td>
			<td></td>
		</tr>
		<tr>
			<td>trifluoroacetic acid (TFA)</td>
			<td>Sigma-Aldrich</td>
			<td>8.0826</td>
			<td></td>
		</tr>
	</tbody>
</table>

synthesis of strong adhesive hydrogel, gelatin o-nitrosobenzaldehyde

Adhesive materials have become popular biomaterials in the field of biomedical and tissue engineering. In our previous work, we presented a new material - gelatin o-nitrosobenzaldehyde (gelatin-NB) - which is mainly used for tissue regeneration and has been validated in animal models of corneal injury and inflammatory bowel disease. This is a novel hydrogel formed by modifying biological gelatin with o-nitrosobenzaldehyde (NB). Gelatin-NB was synthesized by activating the carboxyl group of NB-COOH and reacting with gelatin through 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The obtained compound was purified to generate the final product, which can be stably stored for at least 18 months. NB has a strong adhesion to -NH2 on the tissue, which can form many C = N bonds, thus increasing the adhesion of gelatin-NB to the tissue interface. The preparation process comprises steps for the synthesis of the NB-COOH group, modification of the group, synthesis of gelatin-NB, and purification of the compound. The goal is to describe the specific synthesis process of gelatin-NB in detail and to demonstrate the application of gelatin-NB to damage repair. Moreover, the protocol is presented to further strengthen and expand the nature of the material produced by the scientific community for more applicable scenarios.

Figure 2A shows a schematic of the main chemical reactions involved in the synthesis of gelatin-NB, which promotes tissue integration by grafting NB groups onto gelatin. Figure 2B shows that the O-nitrobenzene of the gelatin-NB hydrogel converts to an NB group immediately after UV irradiation, and then the active aldehyde group can be crosslinked with an amino group to form a Schiff base. Figure 2C indicates that different ratios of...

Watch this Scientific Journal Video about Synthesis of Strong Adhesive Hydrogel, Gelatin O-Nitrosobenzaldehyde at JoVE.com

Synthesis of Strong Adhesive Hydrogel, Gelatin O-Nitrosobenzaldehyde

The protocol presented here shows the synthesis of a strong adhesive hydrogel gelatin o-nitrosobenzaldehyde (gelatin-NB). Gelatin-NB has rapid and efficient tissue adhesion ability, which can form a strong physical barrier to protect wound surfaces, so it is expected to be applied to the field of injury repair biotechnology.

Adhesive materials have become popular biomaterials in the field of biomedical and tissue engineering. In our previous work, we presented a new material - ...

synthesis-of-strong-adhesive-hydrogel-gelatin-o-nitrosobenzaldehyde

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2.2K Views.  Zhejiang University School of Medicine. The protocol presented here shows the synthesis of a strong adhesive hydrogel gelatin o-nitrosobenzaldehyde (gelatin-NB). Gelatin-NB has rapid and efficient tissue adhesion ability, which can form a strong physical barrier to protect wound surfaces, so it is expected to be applied to the field of injury repair biotechnology.

Video: Synthesis of Strong Adhesive Hydrogel, Gelatin O-Nitrosobenzaldehyde

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, 'SurgiLux': Preparation and Demonstration

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures can act as a nidus for infection and in many procedures are unable to effect wound repair or interfere with functional tissue regeneration.1 Surgical glues and adhesives, such as those based on fibrin and cyanoacrylates, have been developed as alternatives to sutures for the repair of such wounds. However, current commercial adhesives also have significant disadvantages, ranging from viral and prion transfer and a lack of repair strength as with the fibrin glues, to tissue toxicity and a lack of biocompatibility for the cyanoacrylate based adhesives. Furthermore, currently available surgical adhesives tend to be gel-based and can have extended curing times which limit their application.2 Similarly, the use of UV lasers to facilitate cross-linking mechanisms in protein-based or albumin 'solders' can lead to DNA damage while laser tissue welding (LTW) predisposes thermal damage to tissues.3 Despite their disadvantages, adhesives and LTW have captured approximately 30% of the wound closure market reported to be in excess of US $5 billion per annum, a significant testament to the need for sutureless technology.4
In the pursuit of sutureless technology we have utilized chitosan as a biomaterial for the development of a flexible, thin film, laser-activated surgical adhesive termed 'SurgiLux'. This novel bioadhesive uses a unique combination of biomaterials and photonics that are FDA approved and successfully used in a variety of biomedical applications and products. SurgiLux overcomes all the disadvantages associated with sutures and current surgical adhesives (see Table 1).
In this presentation we report the relatively simple protocol for the fabrication of SurgiLux and demonstrate its laser activation and tissue weld strength. SurgiLux films adhere to collagenous tissue without chemical modification such as cross-linking and through irradiation using a comparatively low-powered (120 mW) infrared laser instead of UV light. Chitosan films have a natural but weak adhesive attraction to collagen (~3 KPa), laser activation of the chitosan based SurgiLux films emphasizes the strength of this adhesion through polymer chain interactions as a consequence of transient thermal expansion.5 Without this 'activation' process, SurgiLux films are readily removed.6-9 SurgiLux has been tested both in vitro and in vivo on a variety of tissues including nerve, intestine, dura mater and cornea. In all cases it demonstrated good biocompatibility and negligible thermal damage as a consequence of irradiation.6-10

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, &#039;SurgiLux&#039;: Preparation and Demonstration

The fabrication of a novel, flexible thin film surgical adhesive from FDA approved ingredients, chitosan and indocyanine green is described. Bonding of this adhesive to collagenous tissue through a simple activation process with a low-powered infra-red laser is demonstrated.

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Insertion of Flexible Neural Probes Using Rigid Stiffeners Attached with Biodissolvable Adhesive

Microelectrode arrays for neural interface devices that are made of biocompatible thin-film polymer are expected to have extended functional lifetime because the flexible material may minimize adverse tissue response caused by micromotion. However, their flexibility prevents them from being accurately inserted into neural tissue. This article demonstrates a method to temporarily attach a flexible microelectrode probe to a rigid stiffener using biodissolvable polyethylene glycol (PEG) to facilitate precise, surgical insertion of the probe. A unique stiffener design allows for uniform distribution of the PEG adhesive along the length of the probe. Flip-chip bonding, a common tool used in microelectronics packaging, enables accurate and repeatable alignment and attachment of the probe to the stiffener. The probe and stiffener are surgically implanted together, then the PEG is allowed to dissolve so that the stiffener can be extracted leaving the probe in place. Finally, an in vitro test method is used to evaluate stiffener extraction in an agarose gel model of brain tissue. This approach to implantation has proven particularly advantageous for longer flexible probes (&gt;3 mm). It also provides a feasible method to implant dual-sided flexible probes. To date, the technique has been used to obtain various in vivo recording data from the rat cortex.

Insertion of flexible neural microelectrode probes is enabled by attaching probes to rigid stiffeners with polyethylene glycol (PEG). A unique assembly process ensures uniform and repeatable attachment. After insertion into tissue, the PEG dissolves and the stiffener is extracted. An in vitro test method evaluates the technique in agarose gel.

Microelectrode  arrays for neural interface devices that are made of biocompatible thin-film  polymer are expected to have extended functional lifetime ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

Rat Model of Adhesive Capsulitis of the Shoulder

This proposal aims to create an in vivo rat model of adhesive capsulitis for researching potential treatment options for this condition and other etiologies of comparable arthrofibrosis. The model includes extra-articular fixation of the shoulder in rats via scapular to humeral suturing, resulting in a secondary contracture without invading the intra-articular space and resulting in decreased rotational ROM and increased joint stiffness.
We used 10 Sprague-Dawley rats for the purpose of this study. Baseline ROM measurements were taken before glenohumeral immobilization. The rats were subjected to 8 weeks of immobilization before the fixation sutures were removed and changes in ROM and joint stiffness were evaluated. To evaluate whether immobilization resulted in a significant reduction in ROM, changes in kinematics were calculated. ROM was measured at each time point in the follow-up period and was compared to the baseline internal and external ROM measurements. In order to evaluate the stiffness, joint kinetics were calculated by determining the differences in torque (text and tint ) needed to reach the initial external rotation of 60&#176; and initial internal rotation of 80&#176;.
After the removal of the extra-articular suture fixation on follow-up day 0, we found a 63% decrease in total ROM compared to baseline. We observed continuous improvement until week 5 of follow-up, with the progress slowing down around a 19% restriction. On week 8 of follow-up, there was still an 18% restriction of ROM. Additionally, on follow-up day 0, we found the torque increased by 13.3 Nmm when compared to baseline. On week 8, the total torque was measured to be 1.4 &#177; 0.2 Nmm higher than initial measurements. This work introduces a rat model of shoulder adhesive capsulitis with lasting reduced ROM and increased stiffness.

This protocol presents an in vivo rat model of adhesive capsulitis. The model includes an internal fixation of the glenohumeral joint with extra-articular suture fixation for an extended time, resulting in a decreased rotational range of motion (ROM) and increased joint stiffness.

This proposal aims to create an in vivo rat model of adhesive capsulitis for researching potential treatment options for this condition and other ...

Wet-spinning-based Molding Process of Gelatin for Tissue Regeneration

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the wet spinning method, gelatin fibers are produced by smooth extrusion in a suitable coagulation medium. To increase the functional surface of these gelatin fibers and their ability to mimic the features of tissues, gelatin can be molded into a tube form by referring to this concept. Examined by in vitro and in vivo tests, the gelatin tubes demonstrate a great potential for application in tissue engineering. Acting as a suitable filling gap material, gelatin tubes can be used to substitute the tissue in the damaged area (e.g., in the nervous or cardiovascular system), as well as to promote regeneration by providing a direct replacement of stem cells and neural circuitry. This protocol provides a detailed procedure for creating a biomaterial based on a natural polymer, and its implementation is expected to greatly benefit the development of correlative natural polymers, which help to realize tissue regeneration strategies.

We developed and describe a protocol based on the wet spinning concept, for the construction of gelatin-based biomaterials used for the application of tissue engineering.

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...