The authors would like to acknowledge Stanford Department of Orthopaedic Surgery and Stanford Coulter Translational Seed Grant for funding. J.H.L. would like to thank National Science Foundation Graduate Fellowship and DARE Doctoral Fellowship for support.

All experiments were performed in accordance with the Stanford University Human subjects&#8217; guidelines and approved Institutional Review Board protocol.
1. Articular Chondrocyte Isolation
<ol>
	<li>Obtain cartilage from tissue discarded during total knee arthroplasty and dissect as described previously 8.</li>
	<li>Visually select the medial or lateral femoral condyles of cartilage samples for smooth surfaces and make incision at the surface of the cartilage through a scalpel in order to remove 4-5 mm cartilage biopsies without affecting the subchondral bone.</li>
	<li>Isolate chondrocytes from the extracellular matrix by enzymatic dissociation of the tissue with O/N treatment with purified collagenase. Use 1:1 ratio of Collagenase II (125 U/ml) and Collagenase IV (160 U/ml) in chondrocyte growth media at 37 &#176;C.</li>
	<li>The following day, filter the digested tissue containing the dissociated cells through a 70 mm pore size Nylon mesh. Wash two times with 20 ml of DMEM/F12 and centrifuge at 600 x&#160;g for 5 min.</li>
	<li>Plate the isolated chondrocytes at a density of 1 x 106 cells/60 mm culture dish following re-suspension in DMEM/F12 supplemented with 10% Fetal Bovine Serum (FBS), 25 &#181;g/ml&#160;ascorbate, 2 mM L-glutamine, antimicrobials (100 U/ml&#160;penicillin, 100 &#181;g/ml&#160;streptomycin, and 0.25 &#181;g/ml&#160;fungizone). Keep the plates at 37 &#176;C and maintain the primary chondrocyte cultures as a high-density monolayer prior to encapsulation in biomimetic hydrogels.</li>
</ol>
2. Biomimetic Hydrogel Fabrication
NOTE: This method allows the synthesis of a biomimetic hydrogel containing poly(ethylene glycol diacrylate) (PEGDA, MW 5,000 g/mole), chondroitin sulfate-methacrylate (CS-MA) in DPBS. The addition of photoinitiator enables photoactivated crosslinking. The final hydrogel composition contains 7% weight/volume (w/v) of PEGDA, 3% w/v of CS-MA, and 0.05% w/v of photoinitiator.
<ol>
	<li>Synthesize CS-MA by functionalizing CS polymer with methacrylate groups.
	<ol>
		<li>Prepare buffered solution of 50 mM 2-morpholinoethanesulfonic acid (MES) and 0.5 M sodium chloride (NaCl) by dissolving 1.952 g of MES and 5.84 g of NaCl in 200 ml&#160;of deionized water (DiH2O). Dissolve 5 g of chondroitin sulfate sodium salt in the buffered solution.</li>
		<li>Add 0.532 g (4.62 mmol) N-hydroxysuccinimide (NHS) and 1.771 g (9.24 mmol) 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) (molar ratio of NHS:EDC = 1:2) to the solution and stir for 5 min.</li>
		<li>Add 0.765 g (4.62 mmol) 2-aminoethyl methacrylate (AEMA) (molar ratio of NHS:EDC:AEMA = 1:2:1) to the solution and maintain the reaction at RT for 24 hr.</li>
		<li>Purify the mixture by dialysis against deionized water (diH2O) for 4 days using dialysis tubing (12-14 kDa MWCO).</li>
		<li>Filter the purified solution through a 0.22 &#956;m filter and place the open tubes containing the solution in the desiccator and vacuum O/N. Ensure that all tubes are protected from light. Store the dissolved polymer at -20 &#176;C protected from light and moisture.</li>
	</ol>
	</li>
</ol>
3. Cell Encapsulation
<ol>
	<li>Autoclave the PCR film and cylindrical rods (for punching out the cell-laden 3D hydrogels) and sterilize the custom-made cylindrical gel mold by submersion in 0.2 &#181;m filtered 70% ethanol in tissue culture hood under UV light.</li>
	<li>Seal the bottom of the gel mold with autoclaved PCR film without air bubbles or gaps to prevent leaks. Keep it in a sterile 150 mm plate.</li>
	<li>The day prior to cell encapsulation in biomimetic hydrogel, dissociate the high-density chondrocyte monolayer with O/N treatment in Collagenase II and Collagenase IV solution. Use 125 U/ml for Collagenase II and 160 U/ml for Collagenase VI each in chondrocyte growth media at 37 &#176;C.</li>
	<li>Take a 50 ml tube and add 5% weight/volume (w/v) poly(ethylene glycol diacrylate) (PEGDA, MW=5,000 g/mole), 3% w/v chondroitin sulfate-methacrylate (CS-MA), and 0.05% w/v photoinitiator in DPBS. Vortex the tube to mix the solution and keep it aside. Protect the tube from light.</li>
	<li>Collect the dissociated cells in a 50 ml Falcon tube, count and centrifuge at 460 x g for 5 min. Aspirate all the media from the cells tube and add the above mixed gel material to re-suspend cells at a density of 15&#215;106 cells/ml. Mix it 30 times thoroughly while avoiding air bubbles formation.</li>
	<li>Pipette 72 &#181;l of the cell-hydrogel suspension or hydrogel alone into the custom-made cylindrical gel mold and Induce gelation through exposure to UV light (365 nm wavelength) at 3 mW/m2 for 5 min. Prepare some hydrogel solution with no cell content. Use these constructs as negative controls for the future biochemical and mechanical testing. Measure the intensity of the UV light with a UV intensity meter. To adjust intensity, change the distance between the UV light source and the hydrogel scaffold during gelation. 
	NOTE: Since the hydrogel contains CS, the acellular contribution is subtracted from the biochemical GAG content of the cell-laden hydrogels to represent only the cellular GAG.</li>
	<li>Use a scalpel to cut the film for easy peel off and carefully remove it. With the help of the cylindrical rods, push out the gel into a 6 well plate with 5 ml of sterile DPBS to wash off unpolymerized gel and loose cells.</li>
	<li>Culture the cell-laden hydrogels in 24 well plates containing 1.5 ml of chondrocyte growth media into each well. Use a disposable spatula to transfer the washed hydrogels into the well.</li>
	<li>Assess cell viability by live dead staining 24 hr post-encapsulation using a viability/cytotoxity kit. Culture the cell-laden hydrogels and the empty hydrogels for 3-6 weeks changing to fresh chondrocyte growth media every 2 days before harvesting for analyses.</li>
</ol>
4. RNA Extraction and Gene Expression Analyses
<ol>
	<li>Carefully aspirate the media and put sterile PBS 1X on the cell-laden as well as the empty control hydrogels.</li>
	<li>With the help of a sterile spatula, transfer each hydrogel to a clean 1.5 ml eppendorf tube and add 250 &#181;l of Tri-reagent to each tube. Follow manufacturer&#8217;s instructions to isolate RNA.</li>
	<li>After RNA isolation and quantification use 1 &#181;g of it from each sample and perform a reversed transcription into cDNA using the High Capacity cDNA Reverse Transcription Kit.</li>
	<li>For quantitative PCR use TaqMan Gene Expression Arrays for examination of the expression of your genes of interest. Normalize the gene expression levels internally to GAPDH.</li>
	<li>For the PCR conditions include a 2&#160;min incubation at 50 &#176;C to inactivate previous amplicons with uracil-DNA glycosylase, followed by a 10&#160;min incubation at 95 &#176;C to activate the Taq polymerase. Then perform forty cycles of PCR, consisting of 15 sec&#160;at 95 &#176;C, and 1 min&#160;at 60 &#176;C.</li>
</ol>
5. Biochemical Analyses
<ol>
	<li>For each cell-hydrogel constructs quantify DNA and sulfated glycosaminoglycan (sGAG) production as follow.</li>
	<li>Carefully aspirate the media and put sterile PBS 1X on the cell-laden as well as the empty control hydrogels. Weigh an empty tube and put the hydrogel inside after blotting it on tissue paper and record the weight or wet weight of the hydrogels.</li>
	<li>Freeze each hydrogel at -20 &#176;C in a separate 1.5 ml eppendorf tube for enzymatic digestion and later use an aliquot of the digested product to run Picogreen assay (for DNA) and Dimethyl-Methylene blue assay (for GAG).</li>
	<li>Prepare the papainase solution for enzymatic digestion of the hydrogels.
	<ol>
		<li>First prepare the PBE buffer by weighing out 7.1 g of Sodium phosphate dibasic (Na2HPO4) and 1.6 g of Ethylenediaminetetraacetic acid disodium salt (EDTA-Na2). Dissolve in 500 ml of dH2O, adjust the pH to 6.5 and filter the purified solution through a 0.22 mm filter.</li>
		<li>Dissolve 0.035 g of L-cysteine in 20 ml of PBE buffer. Filter the solution and 100 &#181;l of sterile papain enzyme.</li>
	</ol>
	</li>
	<li>When ready to perform the assays, take the tubes from -20 &#176;C and add 300 &#956;l of the ready papain solution to the frozen hydrogels. Crush the gel with a mortar and pestle and mix it well with the motor pestle.</li>
	<li>Bring the volume to 500 &#181;l with additional papain solution. Perform the same procedure for the control gels. Incubate at 60 &#176;C for 16 hr 3,9. The solution containing the digested hydrogel can therefore used for DNA and GAG quantification. Measure the DNA content using the Picogreen assay with Lambda phage DNA as standard following manufacturer&#8217;s protocol.</li>
	<li>Quantify the sulfated-GAG content using the 1,9-dimethylmethylene blue (DMMB) dye-binding assay with shark chondroitin sulfate as standard 10. Determine the GAG content by dividing the amount of GAG for the corresponding wet weight.</li>
</ol>
6. Mechanical Testing
<ol>
	<li>Perform unconfined compression tests using an Instron 5944 testing system fitted with a 10 N load cell.
	<ol>
		<li>After the desired days of in vitro culture, perform the compression test on the cell-hydrogel scaffold and the acellular control hydrogels.</li>
		<li>Submerge the specimens in a PBS bath at RT and compress at a rate of 1% strain/sec to a maximum strain of 15% 11,12 since the physiological strain experienced by cartilage tissue under loading condition has been reported to be 10-20% 13,14.</li>
	</ol>
	</li>
	<li>Create stress vs. strain curves and curve fit using a third order polynomial equation. Determine the compressive tangent modulus from the curve fit equation at strain values of 15%.</li>
</ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+study+of+collagen+types+I+and+II+and+procollagen+IIA+in+human+cartilage+repair+tissue+following+autologous+chondrocyte+implantation.">Immunohistochemical study of collagen types I and II and procollagen IIA in human cartilage repair tissue following autologous chondrocyte implantation.</a> Knee. 16, 398-404 (2009).</li><li>Smeriglio, 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=Comparative+Potential+of+Juvenile+and+Adult+Human+Articular+Chondrocytes+for+Cartilage+Tissue+Formation+in+Three-Dimensional+Biomimetic+Hydrogels.">Comparative Potential of Juvenile and Adult Human Articular Chondrocytes for Cartilage Tissue Formation in Three-Dimensional Biomimetic Hydrogels.</a> Tissue engineering. Part A. , (2014).</li><li>Lai, J. H., Kajiyama, G., Smith, R. 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S., 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=The+effect+of+matrix+stiffness+on+the+differentiation+of+mesenchymal+stem+cells+in+response+to+TGF-beta.">The effect of matrix stiffness on the differentiation of mesenchymal stem cells in response to TGF-beta.</a> Biomaterials. 32, 3921-3930 (2011).</li><li>Wang, T., Lai, J. H., Han, L. H., Tong, X., Yang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrogenic+Differentiation+of+Adipose-Derived+Stromal+Cells+in+Combinatorial+Hydrogels+Containing+Cartilage+Matrix+Proteins+with+Decoupled+Mechanical+Stiffness.">Chondrogenic Differentiation of Adipose-Derived Stromal Cells in Combinatorial Hydrogels Containing Cartilage Matrix Proteins with Decoupled Mechanical Stiffness.</a> Tissue engineering. Part A. , (2014).</li><li>Smith, R. L., 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=Effects+of+intermittent+hydrostatic+pressure+and+BMP-2+on+osteoarthritic+human+chondrocyte+metabolism+in+vitro.">Effects of intermittent hydrostatic pressure and BMP-2 on osteoarthritic human chondrocyte metabolism in vitro.</a> J Orthop Res. 29, 361-368 (2011).</li><li>Buschmann, M. D., Gluzband, Y. A., Grodzinsky, A. J., Kimura, J. H., Hunziker, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrocytes+in+agarose+culture+synthesize+a+mechanically+functional+extracellular+matrix.">Chondrocytes in agarose culture synthesize a mechanically functional extracellular matrix.</a> J Orthop Res. 10, 745-758 (1992).</li><li>Farndale, R. W., Buttle, D. J., Barrett, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+quantitation+and+discrimination+of+sulphated+glycosaminoglycans+by+use+of+dimethylmethylene+blue.">Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue.</a> Biochim Biophys Acta. 883, 173-177 (1986).</li><li>Lai, J. H., Levenston, 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=Meniscus+and+cartilage+exhibit+distinct+intra-tissue+strain+distributions+under+unconfined+compression.">Meniscus and cartilage exhibit distinct intra-tissue strain distributions under unconfined compression.</a> Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society. 18, 1291-1299 (2010).</li><li>Li, L. P., Buschmann, M. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cartilage+stresses+in+the+human+hip+joint.">Cartilage stresses in the human hip joint.</a> Journal of biomechanical engineering. 116, 10-18 (1994).</li><li>Benya, P. D., Shaffer, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dedifferentiated+chondrocytes+reexpress+the+differentiated+collagen+phenotype+when+cultured+in+agarose+gels.">Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels.</a> Cell. 30, 215-224 (1982).</li><li>Buckwalter, J. A., Mankin, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Articular+cartilage:+tissue+design+and+chondrocyte-matrix+interactions.">Articular cartilage: tissue design and chondrocyte-matrix interactions.</a> Instructional course lectures. 47, 477-486 (1998).</li><li>Wang, D. A., 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=Multifunctional+chondroitin+sulphate+for+cartilage+tissue-biomaterial+integration.">Multifunctional chondroitin sulphate for cartilage tissue-biomaterial integration.</a> Nat Mater. 6, 385-392 (2007).</li><li>Simson, J. A., Strehin, I. A., Allen, B. W., Elisseeff, J. 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The authors have nothing to disclose.

As reported in this protocol, 3D hydrogels are able to maintain chondrocyte phenotype in culture, avoiding the process of cell dedifferentiation into fibrocartilage cells usually encountered with monolayer cultures 15. Moreover, long-term cultures of the chondrocytes- hydrogel construct revealed a favorable environment that maintains the intrinsic cell features associated with age and disease.
The use of a 3D biomimetic hydrogel has several advantages. First, the inclusion of chondroitin sulfate (CS), a major component found in articular cartilage, enable cells to degrade the hydrogel matrix by secreting chondroitinase and lay down newly synthesized cartilage extra-cellular matrix 5, 16. In addition, CS has been shown to have anti-inflammatory properties in the arthritic joint. The biomimetic hydrogel may also be used as a scaffolding material for cell delivery in cartilage repair, and may be chemically modified to facilitate better tissue-biomaterial integration 17,18.
The use of the PEG-CS hydrogels allows long-term cultures of chondrocytes and the evaluation of biochemical and mechanical properties. Here we show how this platform can be useful for the comparative analyses of various sources of differentiated chondrocytes in order to define the optimal cell type for cartilage engineering. Interestingly, chondrocytes encapsulated in hydrogels remain viable and proliferate according to their intrinsic abilities. The hydrogel composition supports, in fact, the growth of healthy juvenile and adult chondrocytes as shown in Figure 2. The composition and structure of the described hydrogels also promotes cartilage tissue formation as indicated by the deposition of a functional extracellular matrix assessed by glycosaminoglycan (GAG) quantification.
An additional advantage is that the chondrocyte-hydrogel constructs can be evaluated for the mechanical properties of the newly formed cartilage tissue. Note that the unconfined compression test should be performed on the acellular hydrogel for comparison. The hydrogels, in fact, have an intrinsic stiffness due to the rigidity of the CS moieties. Unconfined compression strain of 5-20% (at a strain rate 1%/s) can be applied for the mechanical testing of cartilage tissue 11,12 since the physiological strain experienced by cartilage tissue under loading condition has been reported to be 10-20% 13,14. The response of both cell-laden and acellular hydrogels to mechanical testing was evaluated at the culture end-point. In the described example above we observed a comparable stiffness of the constructs containing adult and juvenile chondrocytes in contrast to the lower stiffness of the constructs containing OA chondrocytes. Such mechanical properties of the cell-hydrogel construct allow the assessment of the functional properties of the formed tissue giving an in-depth analysis of the cell maturation ability.
In conclusion, the use of the 3D biomimetic hydrogels to study the potential of different chondrocyte population to generate cartilage tissue can be widely applied. Besides the in vitro studies described here, in vivo transplantation of the cell-laden constructs can be envisioned to study cell maturation and regenerative potential in the physiological context. Further modifications of the hydrogel platform with additional biomimetic factors can also be envisioned to optimize chondrocyte proliferation and maturation.

With its limited self-repair potential, human articular cartilage undergoes frequent irreversible damages. Extensive efforts are currently focused on the development of efficient cell-based approaches for treatment of articular cartilage injuries. The success of these cell-based therapies is highly dependent on the selection of an optimal cell source and the maintenance of its regenerative potential. Chondrocytes are a common cell source for cartilage repair, but they are limited in supply and can de-differentiate during in vitro expansion in 2D monolayer culture thereby limiting their generation of hyaline cartilage 1. 
The aim of this protocol is to establish a 3-dimensional hydrogel platform for an in vitro comparative study of human chondrocytes from different ages and disease state. Unlike conventional two-dimensional (2D) culture, three-dimensional (3D) hydrogels allow chondrocytes to maintain their morphology and phenotype and provides a physiologically relevant environment enabling chondrocytes to produce cartilage tissue 2,3. In addition to providing a 3D physical structure for chondrocyte culture, hydrogels mimic the function of native cartilage extracellular matrix (ECM). Specifically, the inclusion of chondroitin sulfate methacrylate provides a potential reservoir for secreted paracrine factors 4 and enables cell-mediated degradation and matrix turnover 5. Although many 3D hydrogel culture systems have been utilized widely in various studies including agarose and alginate gels, we have used a biomimetic 3D culture system that has some distinct advantages for chondrocyte culture. Chondroitin sulfate (CS) is an abundant component in articular cartilage and the PEG-CS hydrogels have been shown to maintain and even enhance chondrogenic phenotype and facilitate cell-mediated matrix degradation and turnover 2,5. In addition, the mechanical properties of the hydrogel scaffold can be easily modulated by changing concentration of PEG and hence can be utilized to further enhance the regeneration potential of chondrocytes or a related cell type 6,7. PEG/CSMA is also biocompatible and hence has the potential for a direct clinical application in cartilage defects for example. The limitation for this system is its complexity and the use of photopolymerization that can potentially affect cell viability as compared to simpler systems like agarose, however the advantages for the chondrocyte culture outweigh the potential limitations.
The 3D hydrogel culture is compatible with conventional assay for evaluation of cell phenotype (gene expression, protein immunostaining) and functional outcome (quantification of cartilage matrix production, mechanical testing). This favorable 3D environment was tested to compare the tissue regeneration potential of human chondrocytes from three different aged populations in long-term 3D cultures. 
The outcomes were evaluated via both phenotypic and functional assays. Juvenile, adult and OA chondrocytes showed differential responses in the 3D biomimetic hydrogel culture. After 3 and 6 weeks, chondrogenic gene expression was upregulated in juvenile and adult chondrocytes but was downregulated in OA chondrocytes. Deposition of cartilage tissue components including aggrecan, type II collagen, and glycosaminoglycan (GAG) was high for juvenile and adult chondrocytes but not for OA chondrocytes. The compressive moduli of the resulting cartilage constructs also exhibited similar trends. In conclusion, both juvenile and adult chondrocytes exhibited chondrogenic and cartilage matrix disposition up to 6 weeks of 3D culture in hydrogels. In contrast, osteoarthritic chondrocytes revealed a loss of cartilage phenotype and minimal ability to generate robust cartilage.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>juvenile chondrocytes (Clonetics&#8482; Normal Human Chondrocyte Cell System )</td>
			<td>Lonza</td>
			<td>CC-2550</td>
			<td></td>
		</tr>
		<tr>
			<td>adult chondrocytes (Clonetics&#8482; Normal Human Chondrocyte Cell System)</td>
			<td>Lonza</td>
			<td>CC-2550</td>
			<td></td>
		</tr>
		<tr>
			<td>poly(ethylene glycol diacrylate)</td>
			<td>Laysan Bio</td>
			<td>ACRL-PEG-ACRL-1000-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>2-morpholinoethanesulfonic acid</td>
			<td>Sigma</td>
			<td>M5287</td>
			<td></td>
		</tr>
		<tr>
			<td>photoinitiator</td>
			<td>Irgacure&#160;</td>
			<td align="right">2959</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium chloride&#160;</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>chondroitin sulfate sodium salt&#160;</td>
			<td>Sigma</td>
			<td>C9819</td>
			<td></td>
		</tr>
		<tr>
			<td>N-hydroxysuccinimide&#160;</td>
			<td>Sigma</td>
			<td align="right">130672</td>
			<td></td>
		</tr>
		<tr>
			<td>1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride</td>
			<td>Sigma</td>
			<td>E1769</td>
			<td></td>
		</tr>
		<tr>
			<td>2-aminoethyl methacrylate</td>
			<td>Sigma</td>
			<td align="right">516155</td>
			<td></td>
		</tr>
		<tr>
			<td>dialysis tubing</td>
			<td>Spectrum Laboratories&#160;</td>
			<td align="right">132700</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase 2</td>
			<td>Worthington Biochemical&#160;</td>
			<td>LS004177</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase 4</td>
			<td>Worthington Biochemical&#160;</td>
			<td>LS004189</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 media</td>
			<td>HyClone, Thermo Scientific&#160;</td>
			<td>SH3002301&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>live/dead assay</td>
			<td>Life Technologies</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/L3224">L3224</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Tri reagent</td>
			<td>Life Technologies</td>
			<td>AM9738</td>
			<td></td>
		</tr>
		<tr>
			<td>Quant-iT&#8482; PicoGreen&#174; dsDNA Assay Kit</td>
			<td>Invitrogen</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/P11496">P11496</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic&#160;</td>
			<td>Sigma</td>
			<td>S3264</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid disodium salt&#160;</td>
			<td>Sigma</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma</td>
			<td>C1276</td>
			<td></td>
		</tr>
		<tr>
			<td>1,9-dimethylmethylene blue&#160;</td>
			<td>Sigma</td>
			<td align="right">341088</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UV light equipment - XX-15LW Bench Lamp, 365nm</td>
			<td>UVP</td>
			<td>95-0042-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Instron 5944 testing system&#160;</td>
			<td>Instron Corporation</td>
			<td>E5940</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Bioactive hydrogels containing PEG and CS moieties (Figure 1) represent an ideal platform for culture and maturation of human articular chondrocytes 2,3,5,7. Chondrocytes from different ages and disease states can be cultured with the described method and analyzed for their phenotype, gene expression and biochemical and mechanical properties of the cartilage tissue generated. Three chondrocyte samples, juvenile- 6 months, adult- 34 years and osteoarthritic- 74 years, were harvested after 3 weeks of culture in 3D biomimetic hydrogels. Gene expression analyses of normal chondrocytes, both juvenile and adult, showed an increase in the expression of the chondrocyte genes Col2a1 and Col6a1. On the contrary, diseased chondrocytes showed a dramatic decrease in Col2a1 while maintaining the expression of Col6a1 (Figure 2) showing a loss of chondrogenic phenotype despite being cultured in a favorable biomimetic environment.
The CS-PEG hydrogels also serve as a dynamic environment for the chondrocytes to proliferate, digest the pre-existing matrix of PEG and CS and deposit their pericellular and extracellular matrix, the main components of the mature cartilage tissue. Given these abilities, chondrocyte expansion after 3 weeks of in vitro culture can be estimated by quantification of DNA with the Picogreen dye. Comparative analysis of the three groups of cells show that the cell density of juvenile and adult populations was unchanged while OA chondrocytes exhibited a dramatic decrease compared to day 1 of culture. A loss of DNA content was also observed for OA but not other chondrocytes suggesting possible cell death during the long-term culture (Figure 3). The secreted matrix was quantified as sulfated-GAG content by DMMB dye-binding assay at the end point stage after 3 weeks of cultures. GAG content is normalized by the wet weight of the hydrogel as recorded before the enzymatic digestion and the acellular contribution is subtracted. As shown in Figure 4, chondrocytes deposited a similar significant amount of GAG during 3 weeks of culture in 3D hydrogels.
The presence of a physical scaffold allows the evaluation of biomechanical properties of the samples through unconfined compression tests 2,3. While acellular hydrogels undergo a decrease in the compressive moduli, cell-laden constructs maintained the compressive moduli after 3 weeks in culture (Figure 4). The phenotypic, biochemical and biomechanical analyses described here are therefore useful for evaluating and understanding the potential of different chondrocyte populations for engineering cartilage.
<img alt="Figure 1" src="/files/ftp_upload/53085/53085fig1.jpg" /> 
Figure 1.&#160;PEG-CS biomimetic hydrogels for 3D chondrocyte culture.&#160;Chondrocytes are resuspended in a mixture containing Poly(ethylene glycol diacrylate) (PEGDA) and chondroitin sulfate-methacrylate (CS-MA) and casted into the custom-made cylindrical gel mold. After UV exposure, solidified gels are collected from the molds and cell viability is assessed by live dead staining 24 hr post-encapsulation. <a href="https://www.jove.com/files/ftp_upload/53085/53085fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53085/53085fig2.jpg" /> 
Figure 2.&#160;Expression of chondrogenic genes in human chondrocytes cultured in 3D biomimetic hydrogels.&#160;Quantitative gene expression of cartilage markers Col2a1 and Col6a1 by juvenile, adult and OA chondrocytes after 3 weeks of culture in 3D biomimetic hydrogels. Values are normalized to gene expression level at day 1. Error bars represent mean&#177; SD. p* &#60;0.05 as determined by a two-tailed Student t test. Modified from Smeriglio et al. 2 <a href="https://www.jove.com/files/ftp_upload/53085/53085fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53085/53085fig3.jpg" /> 
Figure 3.&#160;DNA content in human chondrocytes cultured in 3D biomimetic hydrogels.&#160;DNA quantification by Picogreen assay in juvenile, adult and OA chondrocytes after 3 weeks of culture in 3D biomimetic hydrogels. Values are normalized to DNA level at day 1. <a href="https://www.jove.com/files/ftp_upload/53085/53085fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53085/53085fig4.jpg" /> 
Figure 4.&#160;GAG content measurement and unconfined compression test of human chondrocytes at day 1 and after 3 weeks of culture in 3D biomimetic hydrogels.&#160;(A) GAG quantification by DMMB assay in chondrocytes at day 1 and after 3 weeks of culture in 3D biomimetic hydrogels. Values are normalized to wet weight (w.w.) and expressed as &#181;g/g. (B) Compressive modulus (kPa) of acellular and cell-laden hydrogels at day 1 and after 3 weeks of culture in 3D biomimetic hydrogels. <a href="https://www.jove.com/files/ftp_upload/53085/53085fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about 3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation at JoVE.com

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

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 ...

3d-hydrogel-scaffolds-for-articular-chondrocyte-culture-cartilage

Research

JoVE Journal

Bioengineering

19.9K visualizações.  Stanford University. 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. 

Vídeo: 3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

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

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

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

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

An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular cartilage impairs its load-bearing function substantially as it experiences tremendous chemical degradation, i.e. proteoglycan loss and collagen fibril disruption. One promising way to investigate chemical damage mechanisms during OA is to expose the cartilage specimens to an external solute and monitor the diffusion of the molecules. The degree of cartilage damage (i.e. concentration and configuration of essential macromolecules) is associated with collisional energy loss of external solutes while moving across articular cartilage creates different diffusion characteristics compared to healthy cartilage. In this study, we introduce a protocol, which consists of several steps and is based on previously developed experimental micro-Computed Tomography (micro-CT) and finite element modeling. The transport of charged and uncharged iodinated molecules is first recorded using micro-CT, which is followed by applying biphasic-solute and multiphasic finite element models to obtain diffusion coefficients and fixed charge densities across cartilage zones.

We propose a protocol to investigate the transport of charged and uncharged molecules across articular cartilage with the aid of recently developed experimental and numerical methods.

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread chondrocyte death, potentially leading to irreversible breakdown of the joint and the onset of osteoarthritis. Additionally, maintenance of chondrocyte viability is important in osteochondral graft procedures for optimal surgical outcomes. We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine synovial joints after application of controlled mechanical loads or impacts. This method can be used in comparative studies to investigate the effects of different mechanical loading regimens, different environmental conditions or genetic manipulations, as well as different stages of cartilage degeneration on short- and/or long-term vulnerability of in situ articular chondrocytes. The goal of the protocol introduced in the manuscript is to assess the spatial extent of cell injury/death on the articular surface of murine synovial joints. Importantly, this method enables testing on fully intact cartilage without compromising native boundary conditions. Moreover, it allows for real-time visualization of vitally stained articular chondrocytes and single image-based analysis of cell injury induced by application of controlled static and impact loading regimens. Our representative results demonstrate that in healthy cartilage explants, the spatial extent of cell injury depends sensitively on load magnitude and impact intensity. Our method can be easily adapted to investigate the effects of different mechanical loading regimens, different environmental conditions or different genetic manipulations on the mechanical vulnerability of in situ articular chondrocytes.

We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine joints after application of controlled mechanical loads or impacts. This method can be used to investigate how osteoarthritis, genetic factors and/or different loading regimens affect the vulnerability of in situ chondrocytes.

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread ...

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 ...

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 ...