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 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.
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.
Som rapportert i denne protokollen, 3D hydrogeler er i stand til å opprettholde kondrocytt fenotype i kultur, unngår den type av celle dedifferentiation inn Fibrocartilage celler vanligvis påtreffes med monolagskulturer 15. Videre langsiktige kulturer i chondrocytes- hydrogel konstruere avdekket en gunstig miljø som holder den indre cellefunksjoner knyttet til alder og sykdom.
Bruken av en 3D-biomimetisk hydrogel har flere fordeler. Først, inkludering av chondroitin-sulfat (CS), en hovedkomponent som finnes i leddbrusk, aktiverer cellene til å degradere hydrogelmatriks ved å skille kondroitinase og legge ned nysyntetiserte brusk ekstracellulære matriks 5, 16. I tillegg har CS vist å ha anti-inflammatorisk egenskaper i leddgikt felles. Den biomimetisk hydrogel kan også brukes som en stillasmateriell for celle levering i bruskreparasjon, og kan være kjemisk modifisertå legge til rette for bedre vev-biomateriale integrering 17,18.
Bruken av PEG-CS hydrogeler muliggjør langtidskulturer av kondrocytter og evaluering av biokjemiske og mekaniske egenskaper. Her viser vi hvordan denne plattformen kan være nyttig for de komparative analyser av ulike kilder til differensierte chondrocytes for å definere optimal celletype for brusk engineering. Interessant, kondrocytter innkapslet i hydrogeler forblir levedyktige og proliferere i henhold til deres iboende egenskaper. Hydrogelmaterialet bærere, faktisk, veksten av friske unge og voksne kondrocytter som vist i figur 2. Sammensetningen og strukturen av de beskrevne hydrogeler også fremmer bruskvev dannelse som angitt ved avsetning av en funksjonell ekstracellulær matriks bedømt ved glykosaminoglykan (GAG ) kvantifisering.
En ekstra fordel er at kondrocytt-hydrogel konstruksjonerkan evalueres for de mekaniske egenskaper for det nydannede bruskvev. Merk at unconfined kompresjonstest bør utføres på acellulær hydrogel for sammenligning. De hydrogeler, faktisk har en iboende stivhet på grunn av stivheten til CS grupper. Unconfined komprimering stamme av 5-20% (til en tøyning 1% / s) kan brukes for mekanisk testing av bruskvev 11,12 siden den fysiologiske belastningen oppleves av brusk vevet under lasting tilstand har blitt rapportert å være 10-20 % 13,14. Responsen fra både celle-laden og acellulære hydrogelene til mekanisk testing ble evaluert på kultur endepunktet. I det beskrevne eksempel ovenfor, observerte vi en tilsvarende stivhet i de konstruksjoner som inneholder voksne og unge kondrocytter i motsetning til den mindre stivhet av konstruksjonene som inneholder OA kondrocytter. Slike mekaniske egenskapene til celle hydrogel-konstruksjon tillater vurdering av de funksjonelle egenskaper avdannet vev som gir en grundig analyse av cellemodning evne.
Som konklusjon kan bruken av 3D biomimetic hydrogeler å undersøke potensialet til forskjellige kondrocytt befolkning for å generere bruskvev bli mye brukt. Foruten de in vitro studier er beskrevet her, kan in vivo transplantasjon av celle-laden konstruksjoner tenkes å studere cellemodning og regenererende potensial i fysiologisk sammenheng. Ytterligere modifikasjoner av den hydrogel-plattform med flere biomimetic faktorer kan også tenkes å optimalisere kondrocytt proliferasjon og modning.
The authors have nothing to disclose.
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.
juvenile chondrocytes (Clonetics™ Normal Human Chondrocyte Cell System ) | Lonza | CC-2550 | |
adult chondrocytes (Clonetics™ Normal Human Chondrocyte Cell System) | Lonza | CC-2550 | |
poly(ethylene glycol diacrylate) | Laysan Bio | ACRL-PEG-ACRL-1000-1g | |
2-morpholinoethanesulfonic acid | Sigma | M5287 | |
photoinitiator | Irgacure | 2959 | |
sodium chloride | Sigma | S9888 | |
chondroitin sulfate sodium salt | Sigma | C9819 | |
N-hydroxysuccinimide | Sigma | 130672 | |
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride | Sigma | E1769 | |
2-aminoethyl methacrylate | Sigma | 516155 | |
dialysis tubing | Spectrum Laboratories | 132700 | |
Collagenase 2 | Worthington Biochemical | LS004177 | |
Collagenase 4 | Worthington Biochemical | LS004189 | |
DMEM/F12 media | HyClone, Thermo Scientific | SH3002301 | |
live/dead assay | Life Technologies | L3224 | |
Tri reagent | Life Technologies | AM9738 | |
Quant-iT™ PicoGreen® dsDNA Assay Kit | Invitrogen | P11496 | |
Sodium phosphate dibasic | Sigma | S3264 | |
Ethylenediaminetetraacetic acid disodium salt | Sigma | E5134 | |
L-Cysteine | Sigma | C1276 | |
1,9-dimethylmethylene blue | Sigma | 341088 | |
Instruments | |||
UV light equipment – XX-15LW Bench Lamp, 365nm | UVP | 95-0042-07 | |
Instron 5944 testing system | Instron Corporation | E5940 |