This work was sponsored by the Medicine and Health Technology Plan of Zhejiang Province (2019KY050), the Traditional Chinese Medicine Science and Technology Plan of Zhejiang Province (2019ZA026), the Key Research and Development Plan in Zhejiang Province (Grant No.2020C03043), the Traditional Chinese Medicine Science and Technology Plan of Zhejiang Province (2021ZQ021), and the Zhejiang Provincial Natural Science Foundation of China (LQ22H060007).

This study was approved by the Ethics Committee of Tongde Hospital of Zhejiang Province.
1. Preparation of the DC-ECM hydrogel
NOTE: In this study, the cartilage was obtained from the knee joints of 12 month old Bama miniature pigs, avoiding the collection of bone tissue.
<ol>
	<li>Take the collected cartilage, and block and chop it into 1-2 mm3 pieces with a scalpel.</li>
	<li>Place 20 g of the minced cartilage in a 50 mL centrifuge tube, and add 20 mL of hypotonic Tris-HCl buffer (10 mM Tris-HCl, pH 8.0) to submerge the tissue completely. Place the centrifuge tube in a &#8722;80 &#176;C freezer for 3 h and then in a 37 &#176;C oven for 3 h. Repeat the freezing and heating step six times. In this step, the hypotonic Tris-HCl buffer does not need replacement.</li>
	<li>Shake the centrifuge tube for 30 s using a vortex mixer at a speed of 1,000 rpm. Place a stainless-steel sieve (pore size: ~250 &#181;m) on a new 50 mL centrifuge tube.</li>
	<li>Slowly decant the decellularized cartilage into the stainless-steel sieve, wash the tissue three times with sterile PBS, and collect the cartilage.</li>
	<li>Add 10 mL of trypsin solution (0.25% trypsin in PBS), and place the tube on a shaker at 37 &#176;C for 24 h. During this period, replace the trypsin every 4 h. Filter the suspension using the stainless-steel sieve, and preserve the tissue. The trypsin can be treated overnight during one of the digestion processes, and this will not affect the final digestion.</li>
	<li>Wash the trypsinized tissue with hypertonic buffer (1.5 M NaCl, 50 mM Tris-HCl, pH 7.6).</li>
	<li>Add 10 mL of nuclease solution (50 U/mL deoxyribonuclease and 1 U/mL ribonuclease in 10 mM Tris-HCl, pH 7.5), and place the tube on a shaker at 37 &#176;C for 4 h.</li>
	<li>Remove the nuclease solution, wash the cartilage three times with sterile PBS, and add hypotonic Tris-HCl solution. Place the centrifuge tube on a shaker, and rinse for 20 h at room temperature (RT).</li>
	<li>Remove the hypotonic Tris-HCl solution, wash the cartilage three times with sterile PBS, and add 1% Triton X-100 solution to submerge the tissue for 24 h.</li>
	<li>Remove the Triton X-100 solution, and thoroughly rinse the decellularized cartilage with distilled water for 3 days, changing the distilled water every 12 h.</li>
	<li>Soak the cartilage in peracetic acid solution (0.1% PAA in 4% ethanol) for 4 h. Remove the peracetic acid solution, and wash the cartilage three times with sterile distilled water.</li>
	<li>Place the stainless-steel sieve (pore size: ~250 &#181;m) on a 50 mL centrifuge tube. Slowly decant the decellularized cartilage into the stainless-steel sieve, and collect the cartilage. Test the degree of decellularization and the matrix retention of the cartilage by estimating the DNA, collagen, and glycosaminoglycan (GAG) content.</li>
	<li>Put the decellularized cartilage into a grinding bowl, add liquid nitrogen, and grind the decellularized cartilage to form a powder. Take 2 g of the decellularized cartilage powder, add 80 mL of 0.5 M acetic acid and 20 mg of pepsin, and digest for 24 h. Centrifuge at 400 x g for 10 min; discard the sediment, and collect the supernatant solution (DC-ECM solution).</li>
	<li>Decant 1 mL of DC-ECM solution per well into 6-well plates. Place the 6-well plates in a lyophilizer. Freeze the DC-ECM solution. Once the temperature in the freeze-dryer drops to &#8722;40 &#176;C, turn on the vacuum pump, and maintain the vacuum degree at 10 Pa for 22 h.</li>
	<li>Take out the freeze-dried DC-ECM solution, place it in centrifuge tubes, and store at &#8722;20 &#176;C. The minimum storage period of freeze-dried DC-ECM solution exceeds half a year.</li>
	<li>Add 1 mL of sterile distilled water to dissolve 20 mg of freeze-dried DC-ECM solution in the centrifuge tube. Shake for 1 min using a vortex mixer at a speed of 1,000 rpm at RT. Add 1 mg of vitamin B2 (0.1% w/v) to the DC-ECM solution, incubate at 37 &#176;C for 60 min, and irradiate with UV light (370 nm, 3.5 mW/cm2) for 3 min to form a hydrogel (DC-ECM hydrogel).</li>
</ol>
2. Detection of decellularized cartilage
<ol>
	<li>DNA content detection of decellularized cartilage 
	NOTE: Extract the DNA using the DNEasy Blood &#38; Tissue Kit.
	<ol>
		<li>First, take 20 mg of DC-ECM cartilage in a centrifuge tube. Add 180 &#181;L of Buffer GTL and 20 &#181;L of Proteinase K by vortex oscillation, and incubate the cartilage at 56 &#176;C for 4 h until it is completely dissolved.</li>
		<li>Shake the centrifuge tube for 15 s using a vortex mixer at a speed of 1,000 rpm at RT. Next, add 200 &#181;L of Buffer GL and anhydrous ethanol, and thoroughly mix by vortex vibration. Centrifuge the samples for 1 min at a speed of 6,000 x g at 4 &#176;C.</li>
		<li>Add 500 &#181;L of Buffer GW1 to the adsorption column, and centrifuge the samples for 1 min at a speed of 12,000 x g at 4 &#176;C. Add 500 &#181;L of Buffer GW2 to the adsorption column, and centrifuge at 20,000 x g at 4 &#176;C for 1 min. Finally, add 50 &#181;L of sterilized water to the adsorption column, and centrifuge for 1 min at a speed of 6,000 x g at 4 &#176;C to collect the DNA solution. Determine the DNA content using a spectrophotometer.</li>
	</ol>
	</li>
	<li>Collagen content detection in the decellularized cartilage
	<ol>
		<li>To detect the collagen content in the decellularized cartilage, use hydroxyproline as a collagen amino acid marker. Acidify 5 mg of decellularized cartilage with 5 mL of 6 M hydrochloric acid at 100 &#176;C for 20 min, and then neutralize it with 5 mL of a 6 M sodium hydroxide solution.</li>
		<li>Calculate the content of hydroxyproline by measuring the absorbance of the sample at 570 nm with a spectrophotometer. Obtain a concentration-absorption linear regression using the standard hydroxyproline sample.</li>
	</ol>
	</li>
	<li>GAG content detection in the decellularized cartilage 
	NOTE: The GAG content in the DC-ECM cartilage was detected using a tissue GAG colorimetric quantitative detection kit. All the reagents below are available in the kit.
	<ol>
		<li>Take 200 mg of DC-ECM cartilage powder, and add 500 &#181;L of Reagent A by vortex oscillation. Incubate the sample at 4 &#176;C for 16 h, and then centrifuge at 14,000 x g for 10 min.</li>
		<li>Take 50 mL of sample solution, and add 50 &#181;L of high-salt solution (Reagent B) and 50 &#181;L of acid solution(Reagent C) by vortex oscillation. Incubate the sample for 10 min.</li>
		<li>Add 750 &#181;L of dye solution (Reagent D) by vortex oscillation, and incubate the sample for 30 min in dark conditions. Centrifuge the sample at 14,000 x g for 10 min, and then discard the supernatant.</li>
		<li>Add 1 &#181;L of cleaning solution (Reagent E), and mix well. Centrifuge the sample at 14,000 x g for 10 min, and discard the supernatant.</li>
		<li>Add 1 &#181;L of dissociation solution (Reagent F) to the sample, and mix well. Incubate the sample for 30 min under dark conditions. Finally, determine the GAG content using a spectrophotometer at 600 nm.</li>
	</ol>
	</li>
	<li>Scanning electron microscope (SEM) and transmission electron microscope (TEM) analysis of the decellularized cartilage
	<ol>
		<li>Compare the decellularized cartilage and normal cartilage tissue. Place the sample in a 2.5% glutaraldehyde solution, incubate at 4 &#176;C overnight and then wash with PBS three times.</li>
		<li>Fix the sample in 1% OsO4 for 1 h, and then wash three times with PBS.</li>
		<li>Dehydrate the sample by sequential immersion in 50%, 70%, 90%, and 100% ethanol, as well as 100% acetone for 15 min each.Place the sample in a mixed solution of hexamethyldisilazane and ethanol (1:1) for 15 min, followed by immersion in pure hexamethyldisilazane for 15 min.</li>
		<li>Place the sample in an HCP-2 dryer, and then dry using liquid nitrogen. Then, coat the fully dried specimen with a thin layer of gold-palladium using sputter coating, and image using SEM. The sputter coating was performed at a power of 120 W for 5 min. The experiment was carried out under the following conditions: a working voltage of 15 kV, and a vacuum degree lower than 5 x 10&#8722;5 Pa.</li>
		<li>For the TEM analysis, place the sample in a mixture of anhydrous acetone and resin (1:1) for 1 h, followed by a mixture of anhydrous acetone and resin (1:3) for 3 h. Then, place the sample in resin overnight.
		<ol>
			<li>Place the sample in embedding medium-filled capsules, and heat at 70 &#176;C for 9 h.</li>
			<li>After embedding, cut the specimen into 70 nm thin slices using an ultrafine microtome, and place on a copper mesh.</li>
			<li>Pipet 20 &#181;L of uranyl acetate staining solution onto the copper mesh, and stain for 15 min at RT. Remove the staining solution by gently washing the mesh twice with distilled water.</li>
			<li>Then, pipet 20 &#181;L of lead citrate staining solution onto the copper mesh, and stain for 15 min at RT. Wash the mesh three times with distilled water.</li>
			<li>Place the copper mesh onto a clean Petri dish lined with filter paper. After air-drying, photograph the sections using TEM. The probe was applied with a downward force of 500 nN, scanned at a rate of 1 Hz, and had an elastic constant (k-value) of 40 Nm-1. The radius of the probe tip was measured to be 8 nm.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Vega, S. L., Kwon, M. Y., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+hydrogels+for+cartilage+tissue+engineering.">Recent advances in hydrogels for cartilage tissue engineering.</a> European Cells &amp; Materials. 33, 59-75 (2017).</li><li>Yang, J., Zhang, Y. S., Yue, K., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-laden+hydrogels+for+osteochondral+and+cartilage+tissue+engineering.">Cell-laden hydrogels for osteochondral and cartilage tissue engineering.</a> Acta Biomaterialia. 57, 1-25 (2017).</li><li>Bejleri, D., Davis, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+extracellular+matrix+materials+for+cardiac+repair+and+regeneration.">Decellularized extracellular matrix materials for cardiac repair and regeneration.</a> Advanced Healthcare Materials. 8 (5), e1801217 (2019).</li><li>Brown, M., Li, J., Moraes, C., Tabrizian, M., Li-Jessen, N. Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+extracellular+matrix:+New+promising+and+challenging+biomaterials+for+regenerative+medicine.">Decellularized extracellular matrix: New promising and challenging biomaterials for regenerative medicine.</a> Biomaterials. 289, 121786 (2022).</li><li>Barbulescu, G. I., 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=Decellularized+extracellular+matrix+scaffolds+for+cardiovascular+tissue+engineering:+Current+techniques+and+challenges.">Decellularized extracellular matrix scaffolds for cardiovascular tissue engineering: Current techniques and challenges.</a> International Journal of Molecular Sciences. 23 (21), 13040 (2022).</li><li>Zhang, W., Du, A., Liu, S., Lv, M., Chen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+progress+in+decellularized+extracellular+matrix-derived+hydrogels.">Research progress in decellularized extracellular matrix-derived hydrogels.</a> Regenerative Therapy. 18, 88-96 (2021).</li><li>Zhu, W., 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=Cell-derived+decellularized+extracellular+matrix+scaffolds+for+articular+cartilage+repair.">Cell-derived decellularized extracellular matrix scaffolds for articular cartilage repair.</a> International Journal of Artificial Organs. 44 (4), 269-281 (2021).</li><li>Li, T., Javed, R., Ao, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenogeneic+decellularized+extracellular+matrix-based+biomaterials+for+peripheral+nerve+repair+and+regeneration.">Xenogeneic decellularized extracellular matrix-based biomaterials for peripheral nerve repair and regeneration.</a> Current Neuropharmacology. 19 (12), 2152-2163 (2021).</li><li>Xia, C., 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=Decellularized+cartilage+as+a+prospective+scaffold+for+cartilage+repair.">Decellularized cartilage as a prospective scaffold for cartilage repair.</a> Materials Science &amp; Engineering C-Materials for Biological Applications. 101, 588-595 (2019).</li><li>Chen, P., 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=Desktop-stereolithography+3D+printing+of+a+radially+oriented+extracellular+matrix/mesenchymal+stem+cell+exosome+bioink+for+osteochondral+defect+regeneration.">Desktop-stereolithography 3D printing of a radially oriented extracellular matrix/mesenchymal stem cell exosome bioink for osteochondral defect regeneration.</a> Theranostics. 9 (9), 2439-2459 (2019).</li><li>Saldin, L. 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The authors have nothing to disclose.

This protocol provides a systematic approach for the preparation of decellularized cartilage extracellular matrix hydrogels that closely mimic the native ECM of cartilage tissue. The protocol involves a combination of physical, chemical, and enzymatic disruption to remove cellular material while preserving the structure and composition of the ECM. The protocol's critical steps include adjusting the decellularization time and methods and ensuring complete decellularization.
Compared to other ex...

Cartilage tissue engineering is a rapidly developing field that seeks to regenerate damaged or diseased cartilage tissue1. One key challenge in this field is the development of biomimetic scaffolds that can support the growth and differentiation of chondrocytes, the cells responsible for producing cartilage2. The ECM of cartilage tissue plays a critical role in regulating the behavior of chondrocytes. DC-ECM is an effective scaffold for tissue engineering applications3.
A number of techniques have been developed to produce DC-ECM from cartilage tissue, including chemical, enzymatic, and physical methods. However, these methods often result in the generation of ECM hydrogels that are insufficiently biomimetic, which limits their potential for use in tissue engineering applications4,5. Thus, there is a need for a more effective method for producing DC-ECM hydrogels.
The development of this technique is important because it can advance the field of tissue engineering by providing a new approach for creating biomimetic scaffolds that can support tissue regeneration and repair. Furthermore, this technique could be easily adapted to produce ECM hydrogels from other tissues, thereby expanding its potential applications.
In the broader body of literature, there has been growing interest in using DC-ECM as a scaffold for tissue engineering applications6. Numerous studies have demonstrated the effectiveness of DC-ECM hydrogels in promoting cell growth and differentiation in various tissues, including cartilage7,8. Therefore, the development of a protocol for producing DC-ECM hydrogels that closely mimic the natural ECM of cartilage tissue is a significant contribution to the field.
The protocol presented in this paper addresses this need by providing a novel method for producing DC-ECM hydrogels that closely mimic the natural ECM of cartilage tissue. The protocol involves decellularizing cartilage tissue, isolating the resulting ECM, and creating a hydrogel by cross-linking the ECM with a biocompatible polymer. The resulting hydrogel has shown promising results in supporting the growth and differentiation of chondrocytes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 M Tris-HCl, pH7.6</td>
			<td>Beyotime</td>
			<td>ST776-100 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M Tris-HCl, pH8.0</td>
			<td>Beyotime</td>
			<td>ST780-500 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>-80 &#176;C Freezer</td>
			<td>Eppendorf</td>
			<td>F440340034</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease</td>
			<td>Aladdin</td>
			<td>D128600-80KU</td>
			<td></td>
		</tr>
		<tr>
			<td>DNEasy Blood &#38;Tissue Kit</td>
			<td>Qiagen</td>
			<td>No. 69506</td>
			<td></td>
		</tr>
		<tr>
			<td>GAG colorimetric quantitative detection kit</td>
			<td>Shanghai Haling</td>
			<td>HL19236.2</td>
			<td></td>
		</tr>
		<tr>
			<td>HCP-2 dryer&#160;</td>
			<td>Hitachi</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop8000</td>
			<td>Thermo Fisher</td>
			<td>N/A</td>
			<td>Spectrophotometer</td>
		</tr>
		<tr>
			<td>PBS (10x)</td>
			<td>Gibco</td>
			<td>70011044</td>
			<td></td>
		</tr>
		<tr>
			<td>Ribonuclease</td>
			<td>Aladdin</td>
			<td>R341325-100 mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigma500</td>
			<td>ZIESS</td>
			<td>N/A</td>
			<td>Scanning electron microscope</td>
		</tr>
		<tr>
			<td>Spectra S</td>
			<td>Thermo Fisher</td>
			<td>N/A</td>
			<td>Transmission electron microscope</td>
		</tr>
		<tr>
			<td>Stainless steel sieve</td>
			<td>SHXB-Z-1</td>
			<td>Shanghai Xinbu</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Beyotime</td>
			<td>P0096-500 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin&#160;</td>
			<td>Gibco</td>
			<td>15050065</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraviolet lamp</td>
			<td>Omnicure 2000</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitamin B2</td>
			<td>Gibco</td>
			<td>R4500-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Shanghai Qiasen</td>
			<td>78HW-1&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

synthesis of decellularized cartilage extracellular matrix hydrogels

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their biocompatibility and ability to mimic natural tissue properties. This protocol aims to produce DC-ECM hydrogels that closely mimic the native ECM of cartilage tissue. The protocol involves a combination of physical and chemical disruption and enzymatic digestion to remove the cellular material while preserving the structure and composition of the ECM. The DC-ECM is cross-linked using a chemical agent to form a stable and biologically active hydrogel. The DC-ECM hydrogel has excellent biological activity, spatial structure, and biological induction function, as well as low immunogenicity. These characteristics are beneficial in promoting cell adhesion, proliferation, differentiation, and migration and for creating a superior microenvironment for cell growth. This protocol provides a valuable resource for researchers and clinicians in the field of tissue engineering. Biomimetic hydrogels can potentially enhance the development of effective tissue engineering strategies for cartilage repair and regeneration.

To prepare a better DC-ECM cartilage hydrogel, we studied and reviewed the previous literature and compared the various decellularization protocols in terms of the decellularization ratio, immunogenicity, and mechanical functionality9.
On this basis, we prepared the DC-ECM cartilage hydrogel and explored the effect of a radially oriented extractive matrix/mesenchymal stem cell exosome bio-ink in treating osteochondral defects. The results showed that the DC-ECM cartilag...

Watch this Scientific Journal Video about Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels at JoVE.com

Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels

This paper introduces a new method for the synthesis of decellularized cartilage extracellular matrix (DC-ECM) hydrogels. DC-ECM hydrogels have excellent biocompatibility and provide a superior microenvironment for cell growth. Therefore, they can be ideal cell scaffolds and biological delivery systems.

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their ...

synthesis-of-decellularized-cartilage-extracellular-matrix-hydrogels

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892 Views.  Tongde Hospital of Zhejiang Province. This paper introduces a new method for the synthesis of decellularized cartilage extracellular matrix (DC-ECM) hydrogels. DC-ECM hydrogels have excellent biocompatibility and provide a superior microenvironment for cell growth. Therefore, they can be ideal cell scaffolds and biological delivery systems.

Video: Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

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 ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their cellular source, such as platelet lysate (PL). When used as treatment, EVs are expected to enter the target cells and effect a response from these. In this research, PL-derived EVs have been studied as a cell-free treatment for osteoarthritis (OA). Thus, a method was set up to label EVs and test their uptake on cartilage explants. PL-derived EVs are labeled with the lipophilic dye PKH26, washed twice through a column, and then tested in an in vitro inflammation-driven&#160;OA model for 5 h after particle quantification by nanoparticle tracking analysis (NTA). Hourly, cartilage explants are fixed, paraffined, cut into 6 &#181;m sections to mount on slides, and observed under a confocal microscope. This allows verification of whether EVs enter the target cells (chondrocytes) during this period and analyze their direct effect.

Here, we present a protocol to label platelet lysate-derived extracellular vesicles to monitor their migration and uptake in cartilage explants used as a model for osteoarthritis.

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their ...

Fabrication and Characterization of Layer-By-Layer Janus Base Nano-Matrix to Promote Cartilage Regeneration

Various biomaterial scaffolds have been developed to guide cell adhesion and proliferation in hopes to promote specific functions for in vitro and in vivo uses. The addition of growth factors into these biomaterial scaffolds is generally done to provide an optimal cell culture environment, mediating cell differentiation and its subsequent functions. However, the growth factors in a conventional biomaterial scaffold are typically designed to be released upon implantation, which could result in unintended side effects on surrounding tissue or cells. Here, the DNA-inspired Janus base nano-matrix (JBNm) has successfully achieved a highly localized microenvironment with a layer-by-layer structure for self-sustainable cartilage tissue constructs. JBNms are self-assembled from Janus base nanotubes (JBNts), matrilin-3, and transforming growth factor beta-1 (TGF-&#946;1) via bioaffinity. The JBNm was assembled at a TGF-&#946;1:matrilin-3:JBNt ratio of 1:4:10, as this has been the determined ratio at which proper assembly into the layer-by-layer structure could occur. First, the TGF-&#946;1 solution was added to the matrilin-3 solution. Then, this mixture was pipetted several times to ensure sufficient homogeneity before the addition of the JBNt solution. This formed the layer-by-layer JBNm, after pipetting several times again. A variety of experiments were performed to characterize the layer-by-layer JBNm structure, JBNts alone, matrilin-3 alone, and TGF-&#946;1 alone. The formation of JBNm was studied with UV-Vis absorption spectra, and the structure of the JBNm was observed with transmission electron microscopy (TEM). As the innovative layer-by-layer JBNm scaffold is formed on a molecular scale, the fluorescent dye-labeled JBNm could be observed. The TGF-&#946;1 is confined within the inner layer of the injectable JBNm, which can prevent the release of growth factors to surrounding areas, promote localized chondrogenesis, and promote an anti-hypertrophic microenvironment.

This protocol describes the assembly of a layer-by-layer Janus base nano-matrix (JBNm) scaffold by adding Janus base nanotubes (JBNts), matrilin-3, and Transforming Growth Factor Beta-1 (TGF-&#946;1) sequentially. The JBNm was fabricated and characterized; additionally, it displayed excellent bioactivity, encouraging cell functions such as adhesion, proliferation, and differentiation.

Various biomaterial scaffolds have been developed to guide cell adhesion and proliferation in hopes to promote specific functions for in vitro and in vivo ...

Comparative Study of Decellularization Techniques for Bovine Lung ECM Hydrogels

Development and Characterization of Decellularized Lung Extracellular Matrix Hydrogels

The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells&#39; natural environment in vitro. However, maintaining the native biochemical content of the ECM, achieving mechanical stability, and comprehending the impact of the decellularization process on the mechanical properties of the ECM hydrogels are challenging. Here, a pipeline for decellularization of bovine lung tissue using two different protocols, downstream characterization of the effectiveness of decellularization, fabrication of reconstituted decellularized lung ECM hydrogels and assessment of their mechanical and cytocompatibility properties were described. Decellularization of the bovine lung was pursued using a physical (freeze-thaw cycles) or chemical (detergent-based) method. Hematoxylin and Eosin staining was performed to validate the decellularization and retention of major ECM components. For the evaluation of residual collagen and sulfated glycosaminoglycan (sGAG) content within the decellularized samples, Sirius red and Alcian blue staining techniques were employed, respectively. Mechanical properties of the decellularized lung ECM hydrogels were characterized by oscillatory rheology. The results suggest that decellularized bovine lung hydrogels can provide a reliable organotypic<span style="font-size: 11pt; font-family: Calibri, sans-serif; color: black; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&#160;alternative to commercial ECM products by retaining most native ECM components. Furthermore, these findings reveal that the decellularization method of choice significantly affects gelation kinetics as well as the stiffness and viscoelastic properties of resulting hydrogels.

Author Spotlight: Optimizing Bovine Lung Decellularization for Organotypic Hydrogels

The protocol elucidates two distinct decellularization methodologies applied to native bovine pulmonary tissues, providing a comprehensive account of their respective characterizations.

The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells&#39; natural ...

Decellularized Rat Spleen Matrix Preparation for Liver Tissue Engineering

Fabrication of Decellularized Spleen Matrix Derived from Rats

Liver transplantation is the primary treatment for end-stage liver disease. However, the shortage and inadequate quality of donor organs necessitate the development of alternative therapies. Bioartificial livers (BALs) utilizing decellularized liver matrix (DLM) have emerged as promising solutions. However, sourcing suitable DLMs remains challenging. The use of a decellularized spleen matrix (DSM) has been explored as a foundation for BALs, offering a readily available alternative. In this study, rat spleens were harvested and decellularized using a combination of freeze-thaw cycles and perfusion with decellularization reagents. The protocol preserved the microstructures and components of the extracellular matrix (ECM) within the DSM. The complete decellularization process took approximately 11 h, resulting in an intact ECM within the DSM. Histological analysis confirmed the removal of cellular components while retaining the ECM's structure and composition. The presented protocol provides a comprehensive method for obtaining DSM, offering potential applications in liver tissue engineering and cell therapy. These findings contribute to the development of alternative approaches for the treatment of end-stage liver disease.

Author Spotlight: Utilization of Decellularized Spleen Matrix for Bioartificial Livers

The decellularized spleen matrix (DSM) holds promising applications in the field of liver tissue engineering. This protocol outlines the procedure for preparing rat DSM, which includes harvesting rat spleens, decellularizing them through perfusion, and evaluating the resulting DSM to confirm its characteristics.

Liver transplantation is the primary treatment for end-stage liver disease. However, the shortage and inadequate quality of donor organs necessitate the ...