Name: synthesis of decellularized cartilage extracellular matrix hydrogels
Uploaded: 2023-07-21T14:10:00.000+00:00
Duration: 8 min 34 s
Description: Watch this Scientific Journal Video about Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels at JoVE.com

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>

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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 existing methods for tissue engineering and regenerative medicine, this protocol offers several advantages. DC-ECM hydrogels have excellent biological activity, spatial structure, and biological induction function, as well as low immunogenicity, and these characteristics are beneficial in promoting cell adhesion, proliferation, differentiation, and migration11. DC-ECM hydrogels can also be used for drug delivery and cartilage defect treatment12.
One modification that can be made to this protocol is the use of different cross-linking agents to enhance the mechanical properties of the hydrogel. For example, nano-metal materials can be used to improve the mechanical properties of the hydrogel13. In addition, the concentration of riboflavin and the exposure times to UV light can be optimized to control the compressive and tensile strengths of the hydrogel.
Despite its many advantages, this technique has some limitations that should be considered. One limitation is that the decellularization process may cause some damage to the ECM, leading to changes in its mechanical properties14. Another limitation is that the decellularization process may not completely remove all antigenic material, leading to a potential immune response15. Furthermore, it is important to note that the protocol described in this paper is specific to cartilage tissue, and other types of tissue may require adjustments to the decellularization method.
In terms of future applications, this protocol can be further optimized to develop DC-ECM hydrogels with different compressive and tensile strengths. Additionally, this technique can be applied to other tissues to develop biomimetic hydrogels for tissue engineering and regenerative medicine applications. Overall, the protocol presented in this paper provides a valuable resource for researchers and clinicians in the field of tissue engineering and has the potential to enhance the development of effective tissue engineering strategies for cartilage repair and regeneration.

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><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 &amp;deg;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 &amp;amp;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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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 cartilage hydrogel had low immunogenicity and could enhance cell migration and promote cartilage repair10.
In recent years, we have optimized the preparation of the DC-ECM cartilage. We prepared the decellularized cartilage using the above experimental steps. The results showed that the DNA content was eliminated in the decellularized cartilage compared with that in native cartilage (p &#60; 0.001, Figure 1A). The hydroxyproline test indicated that the collagen content was similar between the native and decellularized cartilages (p = 0.48, Figure 1B). The DMMB assay showed that glycosaminoglycan was well-retained in the decellularized cartilage compared with the native cartilage (p = 0.68, Figure 1C). Furthermore, SEM and TEM were used to observe the ultrastructure of the DC-ECM (Figure 1D).
To prepare a DC-ECM hydrogel, the freeze-dried DC-ECM solution was solubilized for a final concentration of 2 wt%. Further, the DC-ECM solution was mixed with VB2, followed by UVA (370 nm)-induced cross-linking (Figure 2A). We placed the DC-ECM solution and DC-ECM hydrogel into microcentrifuge tubes (Figure 2B). When the tubes were inverted, the hydrogel in the tubes did not flow to the bottom, which was a sign of gelation. The gelation time for the DC-ECM solution based on collagen self-assembly at 37 &#176;C without a cross-linking agent was approximately 15 min (Figure 2C). The viscosity and dynamic modulus of the DC-ECM solution and hydrogel were tested. We found that the solution viscosity of the DC-ECM hydrogel was higher than that of the DC-ECM solution, and higher shear rates were associated with lower solution viscosity (Figure 2D). In addition, the storage modulus of both the DC-ECM solution and hydrogel was much higher than the loss modulus, indicating that they both had properties of a gel rather than a liquid (Figure 2E). Notably, after cross-linking and freeze-drying, the pore sizes of the DC-ECM solution and DC-ECM hydrogel, measured with SEM, decreased from 137.672 &#181;m to 37.936 &#181;m (p &#60; 0.00195, Figure 2F,G).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64797/64797fig01.jpg" /> 
Figure 1:&#160;Preparation and characterization of the decellularized cartilage. The decellularized cartilage was compared with the native cartilage. (A-C) Quantification of the DNA, collagen, and glycosaminoglycan (GAG) content. n = 5, ***p &#60; 0.001 (Student&#39;s t-test). All the experiments were performed at least three times. (D) Microscopic structures of the native cartilage and decellularized cartilage photographed with SEM and TEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64797/64797fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64797/64797fig02.jpg" /> 
Figure 2: Preparation and characterization of the DC-ECM hydrogel. Under ultraviolet light, the DC-ECM solution and VB2 cross-linked and formed a DC-ECM hydrogel. (A) Molecule structures and synthesis of the DC-ECM hydrogel. The appearance, characterization, and mechanical properties were compared between the DC-ECM solution and DC-ECM hydrogel. (B) The images of the DC-ECM solution and DC-ECM hydrogel in microcentrifuge tubes. (C) The gelation time of the DC-ECM hydrogel and DC-ECM/VB2 hydrogel. n = 3, ***p &#60; 0.001 (Student&#39;s t-test). (D) The viscosity and (E)&#160;dynamic modulus of the DC-ECM solution and DC-ECM hydrogel. (F) Microscopic structures of the DC-ECM solution and hydrogel (low and high magnification). Scale bars = 100 &#181;m. (G) Pore sizes of the DC-ECM solution and hydrogel. n = 5, ***p &#60; 0.001 (Student&#39;s t-test). All the experiments were performed at least three times. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64797/64797fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

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JoVE Journal

Bioengineering

939 Views.  Tongde Hospital of Zhejiang Province. Ons protocol is belangrijk omdat het het mogelijk maakt om ziekten te creëren in hydrogels die sterk lijken op de aard van kraakbeenweefsel. Het belangrijkste voordeel van deze techniek is het vermogen om ziekten te produceren in hydrogels met duidelijke biologische activiteit in ruimtelijke structuur, lage immunogeniciteit en biologische industriële functie. Hak om te beginnen het verzamelde kraakbeen met een scalpel in stukjes van één tot twee kubieke millimeter groot.Dompel in e...

Video: Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. Several commercial extracellular matrix products are available as hydrogels or membranes, but these do not possess tissue-specific biological activity. Because perfusion decellularization is usually not possible with human heart tissue, we developed a 3-step immersion decellularization process. Human myocardial slices procured during surgery are first treated with detergent-free hyperosmolar lysis buffer, followed by incubation with the ionic detergent, sodium dodecyl sulfate, and the process is completed by exploiting the intrinsic DNase activity of fetal bovine serum. This technique results in cell-free sheets of cardiac extracellular matrix with largely preserved fibrous tissue architecture and biopolymer composition, which were shown to provide specific environmental cues to cardiac cell populations and pluripotent stem cells. Cardiac extracellular matrix sheets can then be further processed into a microparticle powder without further chemical modification, or, via short-term pepsin digestion, into a self-assembling cardiac extracellular matrix hydrogel with preserved bioactivity.

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

This protocol describes the complete decellularization of human myocardium while preserving its extracellular matrix components. Further processing of the extracellular matrix results in the production of microparticles and a cytoprotective self-assembling hydrogel.

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. ...

Developmental Biology

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

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 ...

Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ultimately rejection can occur. Creating a functional, autologous bio-artificial heart could solve these challenges. Biofabrication of a heart comprised of scaffold and cells is one option. A natural scaffold with tissue-specific composition as well as micro- and macro-architecture can be obtained by decellularizing hearts from humans or large animals such as pigs. Decellularization involves washing out cellular debris while preserving 3D extracellular matrix and vasculature and allowing &#34;cellularization&#34; at a later timepoint. Capitalizing on our novel finding that perfusion decellularization of complex organs is possible, we developed a more &#34;physiological&#34; method to decellularize non-transplantable human hearts by placing them inside a pressurized pouch, in an inverted orientation, under controlled pressure. The purpose of using a pressurized pouch is to create pressure gradients across the aortic valve to keep it closed and improve myocardial perfusion. Simultaneous assessment of flow dynamics and cellular debris removal during decellularization allowed us to monitor both fluid inflow and debris outflow, thereby generating a scaffold that can be used either for simple cardiac repair (e.g. as a patch or valve scaffold) or as a whole-organ scaffold.

This method enables decellularization of a complex solid organ using a simple protocol based on osmotic shock and perfusion of ionic detergent with minimal organ matrix disruption. It comprises a novel decellularization technique for human hearts inside a pressurized pouch with real-time monitoring of flow dynamics and cellular debris outflow.

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, 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 ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure and healing. Extraction of extracellular matrix from fibrogenic cell cultures in vitro has potential for generation of ECM from human- and potentially patient-specific cell lines, minimizing the presence of xenogeneic epitopes which has hindered the clinical success of some existing ECM products. A significant challenge in in vitro production of ECM suitable for implantation is that ECM production by cell culture is typically of relatively low yield. In this work, protocols are described for the production of ECM by cells cultured within sacrificial hollow fiber membrane scaffolds. Hollow fiber membranes are cultured with fibroblast cell lines in a conventional cell medium and dissolved after cell culture to yield continuous threads of ECM. The resulting ECM fibers produced by this method can be decellularized and lyophilized, rendering it suitable for storage and implantation.

The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes.

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

Biology

Decellularization for the Preparation of Highly Preserved Human Acellular Skin Matrix for Regenerative Medicine

Extracellular matrix (ECM) provides biophysical and biochemical stimuli to support self-renewal, proliferation, survival, and differentiation of surrounding cells due to its content of diverse bioactive molecules. Due to these characteristics, the ECM has been recently considered a promising candidate for the creation of biological scaffolds to boost tissue regeneration. Emerging studies have demonstrated that decellularized human tissues could resemble the native ECM in their structural and biochemical profiles, preserving the three-dimensional (3D) architecture and the content of fundamental biological molecules. Hence, decellularized ECM can be employed to promote tissue remodeling, repair, and functional reconstruction of many organs. Selecting the appropriate decellularization procedure is crucial to obtain acellular tissues that retain the characteristics of the ideal microenvironment for cells. 
The protocol described here provides a detailed step-by-step description of the decellularization method to obtain a reproducible and effective cell-free biological ECM. Skin fragments from patients undergoing plastic surgery were scaled down and decellularized using a combination of sodium dodecylsulfate (SDS), Triton X-100, and antibiotics. To promote the regular and homogeneous transport of the solution through the samples, they were enclosed in embedding cassettes to ensure protection from mechanical insults. After the decellularization procedure, the snow-white color of skin fragments indicated complete and successful decellularization. Additionally, decellularized samples showed an intact and well-preserved architecture. The results suggest that the proposed decellularization method was effective, fast, and reproducible and protected samples from architectural damages.

Decellularized human skin is suitable for tissue regeneration. A major issue of decellularization is the preservation of the native architecture, along with the appropriate content of structural proteins, glycosaminoglycans (GAGs), and growth factors. The method proposed allows fast and effective decellularization, producing decellularized skin with well-preserved native features.

Extracellular matrix (ECM) provides biophysical and biochemical stimuli to support self-renewal, proliferation, survival, and differentiation of ...

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 ...

Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels

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